Fluorocyclopentenylcytosine methods of use

ABSTRACT

The disclosed subject matter provides methods using and kits comprising a compound of formula (I) 
     
       
         
         
             
             
         
       
     
     or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof. The disclosed subject matter further provides a method of treating one or more symptoms of cancer comprising administering to a subject in need thereof a compound of formula (I) and a process for preparing such.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/683,408, filed Aug. 22, 2017, which is a divisional of U.S. patentapplication Ser. No. 15/178,390, filed Jun. 9, 2016; claims priority toU.S. Provisional Application No. 62/173,174, filed Jun. 9, 2015; U.S.Provisional Application No. 62/210,708, filed Aug. 27, 2015; U.S.Provisional Application No. 62/289,801, filed Feb. 1, 2016; and U.S.Provisional Application No. 62/319,369 filed Apr. 7, 2016, the contentsof which are hereby incorporated by reference in the entirety.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 7,405,214 (issued Jul. 29, 2008) discloses a compound offormula (I)

also referred to as RX-3117, fluorocyclopentenylcytosine or4-amino-1-((1 S,4R,5S)-2-fluoro-4,5-dihydroxy-3-hydroxymethyl-cyclopent-2-enyl)-1H-pyrimidin-2-one.U.S. Pat. No. 7,405,214 also discloses an 11-steps total synthesis ofRX-3117 from D-ribose and the synthesis uses an expensive catalyst whichposes a challenge for implementation in plant production.

U.S. Pat. No. 9,150,520 (issued Oct. 6, 2015) discloses a short routefor the preparation of RX-3117 through(3R,4R,6aR)-tert-butyl-(5-fluoro-2,2-dimethyl-6-trityloxymethyl-4,6a-dihy-dro-3aH-cyclopenta[1,3]dioxol-4-yloxy)-diphenyl-silaneto 4-amino-1-(3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-d-ihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one.The synthesis requires the intermediates to be isolated in each step.Thus, the process is unsatisfactory for scaled up production of thefinal product due to time and cost constraints. Therefore, there is aneed to provide an improved process, for example by reducing the numberof steps and/or removing the need to purify each intermediate.

SUMMARY OF THE INVENTION

The present invention is directed to new uses and methods of using thecompound of formula (I). The present invention also provides an improvedprocess to significantly reduce the cost of manufacturing by, amongother things, combining multiple steps without isolation andpurification of the intermediate materials. In addition, the presentinvention provides dosage and exposure levels for using the compound offormula (I) in a subject.

One aspect of the disclosure provides a method of treating a tumor byadministering to a subject in need thereof an oral dosage form having aneffective amount of a compound of formula (I)

or a hydrate, a solvate or a pharmaceutically acceptable salt thereof,at a dosage of about 300-2,000 mg/day.

In embodiments, the compound of formula (I) is administrated as amonohydrate. In other embodiments, the compound of formula (I) isadministrated free of solvates, hydrates and salts.

Embodiments of the method can include administering an oral dosage 5 to7 days per week. Embodiments of the method can include administering anoral dosage 5 to 7 days per week for 4 consecutive weeks or for 3consecutive weeks followed by 1 off-week during which the oral dosageform is not administered.

Embodiments of the method can include a dosing cycle consists of either3 consecutive weeks of treatment followed by 1 off week, or 4consecutive weeks of treatment, and the oral dosage form is administeredfor up to 12 dosing cycles.

Embodiments of the method can include an oral dosage form that providesa C_(max) of about 700-1,100 ng/mL after a single administration.Embodiments of the method can include an oral dosage form that providesan AUC_(0-t) (0-24 hours) of about 8,000-10,000 hr·ng/mL after a singleadministration.

Embodiments of the method can be used to treat tumors, includingtreating pancreatic, bladder or colorectal cancer.

Embodiments of the method can include administering the oral dosage formwith a second agent or anti-tumor agent selected from the groupconsisting of antimetabolites, DNA-fragmenting agents, DNA-crosslinkingagents, intercalating agents, protein synthesis inhibitors,topoisomerase I poisons, topoisomerase II poisons, microtubule-directedagents, kinase inhibitors, polyphenols, hormones, hormone antagonists,death receptor agonists, immune checkpoint inhibitors, anti-programmedcell death 1 (PD-1) receptor antibodies and anti-programmed cell deathligand 1 (PD-L1) antibodies. Embodiments of the method can includeadministering a PD-L1 antibody to the subject. Embodiments of the methodcan include administering PD-1 antibody to the subject. Embodiments ofthe method can include administering a solid oral dosage form. Thesecond agent or anti-tumor agent can be administered in the same oraldosage form or in a separate oral dosage form.

In embodiments, the subject in need thereof is a human subject.

Another aspect of the disclosure provides a method of predictingefficacy of treatment of a subject in need thereof with a compound offormula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof,including the steps of: (i) collecting a sample of tumor cell or tissuefrom the subject; and (ii) measuring the level of UCK2 expression in thetumor cell or tissue; wherein the expression level of UCK2 indicates alikelihood of efficacy of treatment with a compound of formula (I).

Another aspect of the disclosure is a kit for testing potential efficacyof a compound of formula (I) or a hydrate, a solvate, or apharmaceutically acceptable salt in the treatment of tumor, by use of anassay that measures levels of kinases, p53, or UCK2 protein in a tumorcell sample.

Another aspect of the disclosure provides a method of treating one ormore symptoms by administering to a subject in need thereof a compoundof formula (I)

at an amount effective to inhibit methyltransferase and to upregulate atleast one hypomethylated target in the subject. In embodiments, thecompound of formula (I) is administrated as a monohydrate. In otherembodiments, the compound of formula (I) is administrated free ofsolvates, hydrates and salts.

Another aspect of the disclosure provides a for preparing of4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one1H₂O (RX-3117-MH), by convertingtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11) to 4-amino-1-((3 aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) in a continuous process with more than one step withoutisolation of any intermediate.

Embodiments of the method can include dissolvingtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11) in 2-methyl tetrahydrofuran; adding tetra-n-butylammoniumfluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol)(INT12) in a reaction solution; and recovering INT12 in an organicphase.

Embodiments of the recovering INT12 in the organic phase can includewashing the reaction solution with an aqueous solution; separating anaqueous extraction from the organic phase having INT12; washing theaqueous extraction with 2-methyl tetrahydrofuran to extract INT12 fromthe aqueous extraction; and combining the extracted INT12 with theorganic phase having INT12.

Embodiments of the method can include adding triethylamine andmethanesulphonyl chloride in 2-methyl tetrahydrofuran to the INT12 inthe organic phase to form((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13) in a second reaction solution; and recoveringINT13 in DMSO.

Embodiments of the recovering INT13 in DMSO can include adding the DMSOto the second reaction solution with INT13; and removing at least 90%w/w of 2-methyl tetrahydrofuran by distillation.

Embodiments of the method can include adding 2.5 equivalents of cesiumcarbonate and cytosine to the INT13 in DMSO to form4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) in a third reaction solution.

Embodiments of the method can include maintaining reaction temperatureat about 33 to 37° C.

In embodiments, the INT14 has a ratio of N- to O-isomers of over about95:5.

Embodiments of the method can include adding an acid to the thirdreaction solution with INT14 to form 4-amino-1-((1 S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one(RX-3117); washing the RX-3117 with methyl tert-butyl ether and water toform an organic phase and an aqueous phase having RX-3117; and purifyingthe RX-3117 to form RX-3117-MH.

Embodiments of the method can include charging the reaction mixture withmethanol and distilling the reaction mixture to remove acetonide untilless than about 1.0% area of the acetonide is detected prior to thewashing step.

Embodiments of the washing step can include separating the aqueous phasehaving RX-3117 from the organic phase; washing the aqueous phase havingRX-3117 with methyl tert-butyl ether until less than about 0.5% w/wtrityl alcohol is detected in the aqueous phase; adding a basic anionresin to the aqueous phase having RX-3117 to form a slurry; filteringthe slurry to retain a mother liquor; concentrating the mother liquor toform a concentrate; and adding acetonitrile to the concentrate to formpurified RX-3117-MH.

Another aspect of the disclosure provides a continuous process forpreparing 4-amino-1-((3 aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) fromtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11), including the steps of: dissolving the ASM11 in 2-methyltetrahydrofuran; adding tetra-n-butylammonium fluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol)(INT12); adding trimethylamine and methanesulphonyl chloride in 2-methyltetrahydrofuran to the INT12 to form((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13); and adding cesium carbonate and cytosine tothe INT13 to form4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14); wherein the steps are performed in one or more fixed reactorswithout isolation of INT12 or INT13.

Another aspect of the disclosure provides a continuous process forpreparing 4-amino-1-((1 S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one1H₂O (RX-3117-MH) from4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14), including the steps of: reacting the INT14 with an acid to form4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one(RX-3117); washing the RX-3117 with methyl tert-butyl ether and water toform an organic phase and an aqueous phase having RX-3117; separatingthe aqueous phase having RX-3117 from the organic phase; washing theaqueous phase having RX-3117 with methyl tert-butyl ether until lessthan about 0.5% w/w trityl alcohol is detected in the aqueous phase;adding a strongly basic anion resin to the aqueous phase having RX-3117to form a slurry; filtering the slurry to retain a mother liquor;concentrating the mother liquor to form a concentrate; addingacetonitrile to the concentrate to form purified RX-3117-MH; andisolating the purified RX-3117-MH; wherein the steps are performed inone or more fixed reactors.

Another aspect of the disclosure provides a method of inducing apoptosisin a cell by contacting the cell with an effective amount of a compoundof formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

Another aspect of the disclosure provides a method of sensitizing a cellto an apoptotic signal by contacting the cell with an effective amountof a compound of formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

Another aspect of the disclosure provides a method of modulating proteinkinase in a cell by contacting the cell with an effective amount of acompound of formula (I) or a hydrate, a solvate, or a pharmaceuticallyacceptable salt thereof.

Another aspect of the disclosure provides a method of treating non-smallcell lung cancer by the steps of: (i) diagnosing a subject withnon-small lung cancer cell; and (ii) administering to the subject aneffective amount of a compound of formula (I) or a hydrate, a solvate,or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a series of bar graphs showing the effect of RX-3117 (1 μM) onA549, SW1573, SW1573/G- and H460 cells in the G1 phase after 24 hours.At a dose of 1 μM, RX-3117 induced accumulation of A549, SW1573,SW1573/G- and H460 cells in the G1 phase after 24 hours exposure.

FIG. 2 is a series of bar graphs showing the effect of RX-3117 (5×IC₅₀)on A549, SW1573 and SW1573/G- cells in the S-phase after 24 hours and 48hours. At a higher dose of 5×IC₅₀, RX-3117 induced the accumulation ofA549, SW1573 and SW1573/G- cells in the S-phase.

FIG. 3 is a series of western blots showing the effect of increasingconcentrations of RX-3117 on pro-caspase 9 activation in SW1573 cells.RX-3117 decreased pro-caspase 9 in SW1573 cells. Reduction ofpro-caspase 9 indicates activation of caspase and subsequentialapoptosis induction.

FIG. 4 is a series of western blots showing the effect of increasingconcentrations of RX-3117 on pro-caspase 9 activation in A549 cells.RX-3117 decreased pro-caspase 9 in A549 cells.

FIG. 5 is a series of western blots showing the double-strand breaks(DSB) induced by RX-3117 as indicated by biomarker γH2A.X (phospho S139)in SW1573 cells after 48 hours. RX-3117 induces DSB indicated by amarker for DSB phosphor S139 H2A.X after 48 h. C* means DNA damage by 50μM etoposide after 2 days, which served as a positive control.

FIG. 6 is a series of western blots showing cleaved PARP induced byincreasing concentrations of RX-3117 after 24 hours.

FIG. 7 is a series of western blots showing the effect of RX-3117 at 1μM and 5 μM on p53 expression levels in A549 cells. At 1 μM and 5 M,RX-3117 increased p53 expression levels in A549 cell line.

FIG. 8 is a series of western blots showing the effect of RX-3117 at 10μM on Chk1, Chk2, Cdk1, Cdk2 and p-Cdc25C expression levels in SW1573cells after 24 and 48 hours. In SW1573 cells Chk1 is increased after 48h of exposure to 10 μM RX-3117, pCDC25C is decreased and Cdk2 isincreased.

FIG. 9 is a series of western blots showing the effect of increasingconcentrations of RX-3117 on wee1 expression levels in SW1573 cellsafter 24 hours. Increasing concentrations of RX-3117 have an effect onwee1, which is decreased after 24 h in SW1573 cell lines.

FIG. 10 is a bar graph showing the effect of RX-3117 at 5×IC₅₀ on PIstained, apoptotic A549 and SW1573 cells in the sub-G1 phase after 24hours (d1) and 48 hours (d2).

FIG. 11 is a bar graph showing the effect of RX-3117 at 5 μM (for A549)and 10 μM (for SW1573) on Annexin V stained, apoptotic A549 and SW1573cells in the sub-G1 phase after 24 hours (d1), 48 hours (d2), 72 hours(d3) and 96 hours (d4).

FIG. 12 is a graph showing the peak plasma concentration C_(max) (ng/mL)following dose 1 and dose 7.

FIG. 13 is a graph showing the plasma exposure in AUC_(0-t) (ng-h/mL)following dose 1 and dose 7.

FIG. 14 shows structural formulas for Cytidine, Gemcitabine, and thenovel cytidine analog RX-3117.

FIG. 15 is a bar graph showing the radiosensitizing effect pre- orpost-incubation with RX-3117. The gray bar represents control,irradiated A2780 cells with 4 Gy, the black bar represents incubationwith 1 μM RX-3117 for 24 hours after irradiation with 4Gy. Thewhite/gray bar represents 24 hours incubation with 1 μM RX-3117 beforeirradiation with 4Gy. P.E. represents plating efficiency.

FIGS. 16A-16F is a series of graphs showing the radiosensitizing effectof 1 μM RX-3117 (5 μM RX-3117 for SW1573 cells) with different doses ofirradiation using a clonogenic assay. Cells were pre incubated withRX-3117 for 24 h. FIG. 16A: A2780 cells had a dose modifying factor(DMF) of 1.8. FIG. 16B: A549 cells had a DMF of 1.8. FIG. 16C: H460cells showed a poor radiosensitizing effect. FIG. 16D: SW1573 cells hadDMF of 1.5. FIG. 16E: SW1573/G- cells had DMF of 1.4. FIG. 16F:Fractionated irradiation of SW1573 cells for 5 days with 2 Gy at 24 hafter incubation with 1 μM RX-3117.

FIGS. 17A-17B is series of graphs showing the radiosensitizing effect ofRX-3117 on spheroids. FIG. 17A: SW1573, normalized spheroid volume over15 days. Control; 1 μM RX-3117 treated; RT treatment of 2 Gy for 5 days;RT treatment of 2 Gy for 5 days and pre incubated with 1 μM RX-3117.FIG. 17B: A549 spheres, normalized spheroid volume over 15 days.Control; 1 μM RX-3117 treated; RT treatment of 2 Gy during 5 days; RTtreatment of 2 Gy for 5 days and pre incubated for 24 h with 1 μMRX-3117.

FIGS. 18A-18B shows western blot analysis of DNA damage. FIG. 18A: Theexpression of the DSB damage marker γH2A.X in A2780 cells exposed toincreasing concentrations starting from 0.1 μM-10 μM RX-3117 for 48 h.FIG. 18B: Time dependent induction of the DNA damage in SW1573 cells incombination with radiation. C* means DNA damage by 50 μM etoposide after2 days, which served as a positive control.

FIGS. 19A-19B is a series of histograms (A) bar graphs and western blots(B) showing disturbance in cell cycle distribution by RX-3117 andradiation in cell lines. FIG. 19A: Histogram of cell phases of celllines treated with 1 μM RX-3117 for 24 h with or without radiation of 4Gy. Cells were harvested 24 h post treatment. FIG. 19B: Cell cycleprotein analysis with western blot 24 h after drug incubation and 30 minafter irradiation.

FIG. 20 is a series of western blots showing the effect of RX-3117 andradiation on the expression of cell cycle proteins in SW1573 cells.Cells were radiated (RT) in the presence and absence of RX-3117.Expression was measured by western blotting using the Odyssey system.

FIG. 21 shows an overview metabolic pathway of RX-3117.

FIG. 22 shows a schematic of the mechanism of downregulation ofmaintenance DNA methyltransferase (DNMT1) by RX-3117.

FIG. 23 is a series of western blots showing the effect of RX-3117 at 1μM, 5 μM, 25 M, and 75 μM on DNMT1, DNMT3A, DNMT3B, and (3-actinexpression levels in A549 cells. RX-3117 downregulates maintenance ofDNA methyltransferase 1.

FIG. 24 is a diagram showing the potential effects on cell cycleproteins: regulation of cell cycle by checkpoint kinases Chk1 and Chk2after damage induction.

FIGS. 25A-25D is a series of graphs and associated western blots showingthe effect of RX-3117 on A549 and SW1573 cells at 5 M, 10 M, 20 μM and50 μM for 24 or 48 hours.

Cells were harvested and protein expression was measured using westernblotting (FIGS. 25A and 25B). RNA was isolated and gene expression wasmeasured using RT-PCR (FIGS. 25C and 25D). RX-3117 down regulates DNMT1protein and gene expression.

FIGS. 26A-26B is a western blot and a graph showing the effect ofRX-3117 (1 μM) and aza-dC (5 μM) on A2780 ovarian cancer cells for 24hr. Nuclear extracts were isolated and DNMT1 expression was measured bywestern blot (FIG. 26A) and activity (FIG. 26B) by a commercial kit asdescribed in the Methods.

FIGS. 27A-27C shows the effects of RX-3711 and aza-dC on A549 cells.Global methylation was measured using FACS (FIG. 27A) orimmunofluorescence (FIG. 27B) with an antibody against5-methyl-cytosine. Control cells were set at 100% (FIG. 27A). Thewestern blots (FIG. 27C) shows the expression of MGMT and E-cadherin inA549 cells, and p16 in SW1573 cells after exposure to RX-3117 andaza-dC.

FIGS. 28A-28C shows the effect of RX-3117 on PCFT mediated transport ofMTX for 24 hours. Folic acid (FA) was added to inhibit PCFT and L-LV toinhibit RFC mediated MTX transport. Aza-CdR and aza-CR were included asa positive control.

FIG. 29 is a ¹H NMR of RX-3117 made using the process described inExample 9.

FIG. 30 is a ¹³C NMR of RX-3117 made using the process described inExample 9.

FIG. 31 is a ¹⁹F NMR of RX-3117 made using the process described inExample 9.

FIG. 32 is a Mass Spectrum of RX-3117 made using the process describedin Example 9.

FIG. 33 is a Mass Spectrum (with an ES− filter) of RX-3117 made usingthe process described in Example 9.

FIG. 34 is a microscopic comparison of RX-3117 made according to theprocess of Example 9 (Top row) and prepared using a laboratory scaleprocess (bottom row) under plain polarized light (left column) and crosspolarized light (right column.

FIG. 35 is an X-Ray Powder Diffraction data comparing RX-3117 made usinga laboratory scale process (top spectrum) and RX-3117 made using theprocess described in Example 9 (bottom spectrum).

DETAILED DESCRIPTION Definitions

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which the disclosed subject matter belongs. One ofordinary skill in the art will appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the disclosed subject matter.

Unless clearly indicated otherwise, the following terms as used hereinhave the meanings indicated below. These meanings are intended tosupplement, rather than alter, the meanings of these terms as understoodin the art.

“C_(max)” refers to the maximum observed plasma concentration.

“T_(max)” refers to the time at which C_(max) is attained.

“T_(1/2)” refers to the time required for the plasma concentration of adrug to reach half of its original value. “Terminal T_(1/2)” refers toT_(1/2) in the terminal phase.

“AUC_(0-t)” refers to the area under the plasma concentration versustime curve (AUC) from time zero to time t, where “t” is the lastsampling time point with measurable concentration. For example, AUC₀₋₂₄or AUC_(0-t) (0-24 hours) refers to the AUC from time zero to 24 hours.

“Oral dosage form” refers to a pharmaceutical composition formulated fororal administration. The oral dosage form can be formulated to provideimmediate, sustained, extended, delayed or controlled release. Examplesof an oral dosage form include tablets, capsules, granulations andgel-caps.

“Effective amount” refers to an amount of a compound or pharmaceuticalcomposition that, based on its parameters of efficacy and potential fortoxicity and the knowledge of one skilled in the art, produces a desiredeffect, such as treating or preventing a condition. An effective amountcan be administered in one or more doses.

“Contacting” refers to causing, either directly or indirectly, acompound and a cell to be in sufficient proximity to produce a desiredeffect, such as inducing apoptosis or modulating protein kinase. Thecontacting may be performed in vitro or in vivo. For example, contactinga cell with a compound may involve delivering the compound directly intothe cell using known techniques such as microinjection, administeringthe compound to a subject carrying the cell, or incubating the cell in amedium that includes the compound.

“Treating” refers to attaining a beneficial or desired result, such as aclinical result. In some embodiments, the beneficial or desired resultis any one or more of the following: inhibiting or suppressing the onsetor development of a condition, reducing the severity of the condition,reducing the number or severity of symptoms associated with thecondition, increasing the quality of life of a subject suffering fromthe condition, decreasing the dose of another medication required totreat the condition, enhancing the effect of another medication asubject is taking for the condition, and prolonging the survival of asubject having the condition.

“Preventing” refers to reducing the probability that a subject developsa condition which the subject does not have but is at risk ofdeveloping. “At risk” denotes that a subject has one or more riskfactors, which are measurable parameters that correlate with thedevelopment of a condition and are known in the art. A subject havingone or more of risk factors has a higher probability of developing thecondition than a subject without such risk factors.

“Subject” refers to an animal, such as a mammal, including, but notlimited to, a human. Hence, the methods disclosed herein can be usefulin human therapy and veterinary applications. In one embodiment, thesubject is a mammal. In other embodiments, the subject is a human.

“Fasted” refers to a subject that has fasted from food for at least 8hours prior to treatment.

“Apoptosis” or “apoptotic process” refers to a programmed cell deathprocess which begins when a cell receives an internal or external signal(apoptotic signal), and proceeds through a series of biochemical events(signaling pathway phase) which trigger an execution phase. In theexecution phase, effector caspases cleave vital cellular proteinsleading to the morphological changes that characterize apoptosis. Thesechanges can include, for example, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, formation of membranevesicles (apoptotic bodies), deoxyribonucleic acid (DNA) fragmentation,chromatin condensation, chromosome migration, margination in cellnuclei, mitochondrial swelling, widening of the mitochondrial cristae,opening of the mitochondrial permeability transition pores, dissipationof the mitochondrial proton gradient, and/or plasma membrane blebbing.Exemplary assays used to detect and measure apoptosis includemicroscopic examination of pyknotic bodies as well as enzymatic assayssuch as Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL), caspase assay, annexin assay and DNA laddering. Apoptotic cellscan be quantitated, for example, by FACS analysis of cells stained withpropidium iodide for DNA hypoploidy.

“Inducing apoptosis” refers to causing apoptosis directly or indirectly,and may be characterized by an increased number of cells in a given cellpopulation that undergo apoptosis, an increased rate by which a cellundergoes apoptosis, or an increased intensity, number or rate of onsetof one or more morphological characteristics of apoptosis.

“Sensitizing” refers to increasing a cell's sensitivity to, or reducinga cell's resistance in responding to, an apoptotic signal.

“Apoptotic signal” refers to a stimulus that activates an apoptoticsignaling pathway.

“Apoptotic signaling pathway” refers to a series of molecular signalsthat triggers apoptotic cell death. The pathway starts with thereception of a signal, and ends when the execution phase of apoptosis istriggered.

“Modulating” refers to altering the expression and/or activity of abiomolecule such as a protein kinase. In one embodiment, modulatingrefers to increasing the expression and/or activity of a biomolecule. Inother embodiments, modulating refers to decreasing the expression and/oractivity of a biomolecule.

“Protein kinase” refers to a kinase enzyme that modifies other proteinsby phosphorylation. Examples of a protein kinase includeserine/threonine protein kinase (e.g., checkpoint kinase 1, checkpointkinase 2), tyrosine-specific protein kinase, histidine-specific proteinkinase, and mixed kinase (e.g., mitogen-activated protein kinasekinase). In one embodiment, the protein kinase is a serine/threonineprotein kinase. In one embodiment, the protein kinase is aserine/threonine protein kinase. In other embodiments, the proteinkinase is checkpoint kinase 1 (Chk1) or checkpoint kinase 2 (Chk2). Inother embodiments, the protein kinase is Chk1. In other embodiments, theprotein kinase is Chk2.

“p53” refers to a protein encoded by the p53 tumor suppressor gene.

“UCK2” refers to Uridine Cytidine Kinase 2 expressed predominantly intumor cell or tissue.

“Tumor cell” refers to a cell derived from a tumor.

“Tumor” refers to an abnormal growth of tissue or cells, whether benignor malignant. Examples include tumors found in prostate, lung, brain,breast, kidney, liver, lung, intestines, lymph, muscle, bone, bonemarrow, uterus, ovary, vagina, vulva, pancreas, adrenal gland, centralnervous system, peripheral nervous system, cervix, bladder, endometrium,throat, esophagus, larynx, thyroid, blood, penal, testicular, thymus,skin, spine, stomach, bile duct, small bowel, hepatobiliary tract,colorectal, colon, rectum, anus, endocrine, eye, and gall bladder.

“Cancer” refers to a malignant tumor. Cancer cells may or may not invadethe surrounding tissue and, hence, may or may not metastasize to newbody sites. Cancer encompasses carcinomas, which are cancers ofepithelial cells; carcinomas include squamous cell carcinomas,adenocarcinomas, melanomas, and hepatomas. Cancer also encompassessarcomas, which are tumors of mesenchymal origin; sarcomas includeosteogenic sarcomas, leukemias, and lymphomas. Cancers may involve oneor more neoplastic cell type.

“Anti-tumor agent” refers to any agent useful for treating or preventingtumor. Examples of an anti-tumor agent include the active agentsdescribed in Pharmaceutical Compositions, infra. In embodiments, theanti-tumor agent in addition to RX-3117 is selected fromantimetabolites, DNA-fragmenting agents, DNA-crosslinking agents,intercalating agents, protein synthesis inhibitors, topoisomerase Ipoisons, topoisomerase II poisons, microtubule-directed agents, kinaseinhibitors, polyphenols, hormones, hormone antagonists, death receptoragonists, immune checkpoint inhibitors, anti-programmed cell death 1(PD-1) receptor antibodies and anti-programmed cell death ligand 1(PD-L1) antibodies. In other embodiments, the additional anti-tumoragent is a PD-1 receptor antibody. In other embodiments, the additionalanti-tumor agent is pembrolizumab. In other embodiments, the additionalanti-tumor agent is nivolumab. In other embodiments, the additionalanti-tumor agent is duryalumab. In other embodiments, the additionalanti-tumor agent is combination of nivolumab and pembrolizumab.

“Radiation” refers to any radiation useful for treating or preventingtumor. Examples of radiation include X-rays, gamma rays, and chargedparticles. The radiation may be delivered by any form of radiationtherapy, such as external beam radiotherapy (EBRT, XBRT or teletherapy),brachytherapy (internal radiation therapy or sealed source therapy),intraoperative radiotherapy, or systemic radiation therapy.

“Isolation” refers to any process by which an intermediate is separatedfrom a reaction mixture by purification such as by chromatography,distillation, filtration, extraction, drying or recrystallization.

“Fixed reactor or vessel” refers to a reactor system in a fixed place inthe plan that cannot be moved.

“Such as” has the same meaning as “such as but not limited to.”Similarly, “include” has the same meaning as “include but not limitedto,” while “including” has the same meaning as “including but notlimited to.”

The singular forms “a,” “or,” and “the” include plural references unlessthe context dictates otherwise. Thus, for example, a reference to “acompound” may include one or more compound(s) and/or equivalent(s)thereof.

Any numerical range disclosed herein encompasses the upper and lowerlimits and each intervening value, unless otherwise specified.

Other than in the working examples, or where otherwise indicated,numerical values (such as numbers expressing quantities of ingredients,reaction conditions) as used in the specification and claims aremodified by the term “about”. Accordingly, unless indicated to thecontrary, such numbers are approximations that may vary depending uponthe desired properties sought to be obtained. At the very least, and notas an attempt to limit the application of the doctrine of equivalents tothe scope of the claims, each numerical parameter should be construed inlight of the number of significant digits and ordinary roundingtechniques.

While the numerical parameters setting forth the scope of the disclosedsubject matter are approximations, the numerical values set forth in theworking examples are reported as precisely as possible. Any numericalvalue, however, inherently contains certain errors necessarily resultingfrom the standard deviation found in its respective testingmeasurements.

Methods of Inducing Apoptosis or Sensitizing a Cell to an ApoptoticSignal

One aspect of the disclosure provides a method of inducing apoptosis bythe steps of administering an effective amount of a compound of formula(I) (RX-3117)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.In embodiments, the compound of formula (I) may be administrated as amonohydrate or free form.

Another aspect of the disclosure provides a method of inducing apoptosisin a cell by the steps of contacting the cell with an effective amountof a compound of formula (I) or a hydrate, a solvate, or apharmaceutically acceptable salt thereof.

In one embodiment, the method induces apoptosis through single-strandbreak (SSB) or double-strand break (DSB). In other embodiments, themethod induces apoptosis through SSB. In other embodiments, the methodinduces apoptosis through DSB.

Another aspect of the disclosure provides a method of sensitizing a cellto an apoptotic signal by contacting the cell with an effective amountof a compound of formula (I) or a hydrate, a solvate, or apharmaceutically acceptable salt thereof.

In one embodiment of any the methods provided herein, the cell is atumor cell. In other embodiments, the cell is a malignant tumor cell. Inother embodiments, the cell is a lung cancer cell. In other embodiments,the cell is a non-small cell lung cancer cell. In other embodiments, thecell is a pancreatic cancer cell. In other embodiments, the cell is abladder cancer cell. In other embodiments, the cell is a colorectalcancer cell. In other embodiments, the cell is a mammalian cell or acell in a mammal. In other embodiments, the cell is a human cell or acell in a human.

Methods of Modulating Protein Kinase

Another aspect of the disclosure provides a method of modulating aprotein kinase in a cell in which the method includes contacting thecell with an effective amount of a compound of formula (I) or a hydrate,a solvate, or a pharmaceutically acceptable salt thereof. Inembodiments, the protein kinase is modulated by increasing the proteinkinase. The increase can be by, for example, 5% or more, 10% or more,20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 75% or more, 80% or more, 90% or more, or 95% ormore.

In embodiments, the protein kinase is a checkpoint protein kinase. Inother embodiments, the protein kinase is checkpoint kinase 1 (Chk1) orcheckpoint kinase 2 (Chk2). In other embodiments, the protein kinase isChk1. In other embodiments, the protein kinase is Chk2.

In embodiments, the cell is in a mammal. In other embodiments, the cellis in a human.

In embodiments, the method increases the protein kinase expressionlevel.

Methods of Treating or Preventing Non-Small Cell Lung Cancer

Another aspect of the disclosure provides a method of treating orpreventing a non-small cell lung cancer by the steps of:

(i) diagnosing a subject with non-small lung cancer cell; and

(ii) administering to the subject an effective amount of a compound offormula (I) or a hydrate, a solvate, or a pharmaceutically acceptablesalt thereof.

Methods of Predicting Efficacy of Treatment

Another aspect of the disclosure provides a method of predictingefficacy of treatment of a subject in need thereof with a compound offormula (I) or a hydrate, a solvate, or a pharmaceutically acceptablesalt thereof, by the steps of:

(i) collecting a sample of tumor cell or tissue from the subject;

(ii) measuring one of protein kinases or p53 expression level in thesample,

(iii) contacting the tumor cell or tissue with a compound of formula(I);

(iv) measuring one of protein kinases or p53 expression level in thetumor cell or tissue after contact with the compound of formula (I),wherein an increase in protein kinases or p53 expression level indicateslikelihood of efficacy.

In embodiments, contacting the tumor cell or tissue with a compound offormula (I) is accomplished by contacting the sample of tumor cell ortissue. In such embodiments, measuring one of protein kinases or p53expression level in the tumor cell or tissue after contact with thecompound of formula (I) is conducted on the sample. In otherembodiments, contacting the tumor cell or tissue with a compound offormula (I) is accomplished by administering the compound of formula (I)to the subject. In such embodiments, measuring one of protein kinases orp53 expression level in the tumor cell or tissue after contact with thecompound of formula (I) is conducted by collecting a second sample oftumor cell or tissue from the subject and measuring one of proteinkinases or p53 expression level in the second sample.

In embodiments, the method further comprises administering the subjectwith the compound of formula (I) if an increase in one of proteinkinases or p53 expression level is detected. The protein kinase isconsidered to have increased if the amount of protein kinase is greaterby a statistically significant amount. Thus the increase may be by 5% ormore, 10% or more, 20% or more, or 25% or more. In other embodiments,the method further comprises the step of measuring protein Cdc25C orp-Cdc25C expression level in the sample in steps (ii) and (iv), whereina decrease in protein Cdc25C or p-Cdc25C expression level indicateslikelihood of efficacy. The protein Cdc25C or p-Cdc25C expression levelis considered to have decreased if the protein Cdc25C or p-Cdc25Cexpression level is reduced by a statistically significant amount. Thusthe reduction may be by 5% or more, 10% or more, 20% or more, or 25% ormore. In other embodiments, the method further comprises administeringthe subject with the compound of formula (I) if an increase in one ofprotein kinases or p53 expression level and a decrease in protein Cdc25Cor p-Cdc25C expression level are detected.

In embodiments, the compound of formula (I) is administrated as amonohydrate. In other embodiments, the compound of formula (I) isadministrated free of solvates, hydrates and salts.

In embodiments, the method comprises measuring protein kinase expressionlevel in steps (ii) and (iv). In other embodiments, the protein kinaseis a checkpoint kinase. In other embodiments, the protein kinase is Chk1or Chk2. In other embodiments, the protein kinase is Chk1. In otherembodiments, the protein kinase is Chk2.

Another aspect of the disclosure provides a method of predictingefficacy of treatment of a subject in need thereof with a compound offormula (I) or a hydrate, a solvate, or a pharmaceutically acceptablesalt thereof, by the steps of:

(i) collecting a sample of tumor cell or tissue from the subject; and

(ii) measuring the level of UCK2 expression in the tumor cell or tissue;

wherein the expression level of UCK2 indicates a likelihood of efficacyof treatment with a compound of formula (I).

In embodiments, the method further comprises administering the subjectwith the compound of formula (I) if an increased expression of UCK2 ismeasured in the tumor cell or tissue. In embodiments, UCK2 expression ismeasured by immunoblotting the protein level of UCK2 normalized tobeta-actin. The UCK2 is considered to have an increased expression levelif the amount of UCK2 expression is greater by a statisticallysignificant amount compared to a predetermined level. Thus the increasemay be by 5% or more, 10% or more, 20% or more, or 25% or more. Inembodiments, the predetermined level may be the level of UCK2 expressionon a non-tumor cell. In embodiments, the predetermined level may be thelevel of UCK2 expression on a non-tumor cell of the subject.

In embodiments, the subject is a mammal. In other embodiments, thesubject is a human.

In embodiments, the tumor cell is lung cancer cell. In otherembodiments, the tumor cell is non-small cell lung cancer cell.

Methods of Treating or Preventing a Tumor

Another aspect of the disclosure provides a method of treating orpreventing a tumor by the steps of administering to a subject in needthereof an oral dosage form that includes an effective amount of acompound of formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof,at a dosage of about 300-2,000 mg/day. In other embodiments, the dosageis about 400-800 mg/day. In other embodiments, the dosage is about500-700 mg/day. In other embodiments, the dosage is about 300 mg/day. Inother embodiments, the dosage is about 400 mg/day. In other embodiments,the dosage is about 500 mg/day. In other embodiments, the dosage isabout 600 mg/day. In other embodiments, the dosage is about 700 mg/day.In other embodiments, the dosage is about 800 mg/day.

The dosage of about 300-2,000 mg/day is based upon an adult human havinga weight or body mass of about 60-80 kg. Thus, the dosage can range fromabout 5-33 mg/kg/day. Additional dosages based on subject weight may bereadily calculated from these values. Similarly, persons skilled in theart will be able to calculate dosages for other species based on knowncorrelations to human dosages.

In embodiments, the oral dosage form is administered 3-7 days per week.In other embodiments, the oral dosage form is administered 4-7 days perweek. In other embodiments, the oral dosage form is administered 5-7days per week. In other embodiments, the oral dosage form isadministered 5 or 7 days per week. In other embodiments, the oral dosageform is administered 3 days per week. In other embodiments, the oraldosage form is administered 4 days per week. In other embodiments, theoral dosage form is administered 5 days per week. In other embodiments,the oral dosage form is administered 6 days per week. In otherembodiments, the oral dosage form is administered 7 days per week.

In embodiments, the total daily dose is administered in one or moredoses. In other embodiments, the oral dosage form is administered oncedaily. In other embodiments, the oral dosage form is administered twicedaily. In other embodiments, the oral dosage form is administered threetimes daily. In other embodiments, the oral dosage form is administeredfour times daily.

In embodiments, the oral dosage form is administered at a dosage of upto about 20,000 mg/month. The total monthly dose can be administered 1-7days per week either for three weeks followed by one week of rest (“offweek”), or for four weeks without rest. For each week of treatment, theoral dosage form may be administered 1-7 days per week. In oneembodiment, the oral dosage form is administered for three weeksfollowed by one week of rest. In other embodiments, the oral dosage formis administered 3-7 days per week for three weeks followed by one weekof rest. In other embodiments, the oral dosage form is administered 5-7days per week for three weeks followed by one week of rest. In otherembodiments, the oral dosage form is administered daily for three weeksfollowed by one week of rest. In other embodiments, the oral dosage formis administered daily for 28 days. Each dosing cycle consists of either3 weeks of treatment followed by 1 week of rest, or 4continuous/consecutive weeks of treatment. The dosing cycle may berepeated as often as necessary as determined by a person skilled in theart. In one embodiment, the oral dosage form is administered for up to12 dosing cycles. In other embodiments, the oral dosage form isadministered for up to 6 dosing cycles.

In embodiments, the oral dosage form is administered at a dosage ofabout 300-2,000 mg/day 5-7 days per week. In other embodiments, thedosage is about 400-800 mg/day 5-7 days per week. In other embodiments,the dosage is about 500-700 mg/day 5-7 days per week. In otherembodiments, the dosage is about 500-700 mg/day 5 or 7 days per week. Inother embodiments, the dosage is about 500 mg/day 5 days per week. Inother embodiments, the dosage is about 500 mg/day 7 days per week. Inother embodiments, the dosage is about 600 mg/day 5 days per week. Inother embodiments, the dosage is about 600 mg/day 7 days per week. Inother embodiments, the dosage is about 700 mg/day 5 days per week. Inother embodiments, the dosage is about 70 mg/day 7 days per week.

In embodiments, the oral dosage form is administered once daily at about400 mg/day 5 days per week. In other embodiments, the oral dosage formis administered once daily at about 500 mg/day 5 days a week. In otherembodiments, the oral dosage form is administered once daily at about600 mg/day 5 days per week. In other embodiments, the oral dosage formis administered once daily at about 700 mg/day 5 days per week. In otherembodiments, the oral dosage form is administered once daily at about800 mg/day 5 days per week.

In embodiments, the oral dosage form is administered once daily at about400 mg/day 7 days per week. In other embodiments, the oral dosage formis administered once daily at about 500 mg/day 7 days per week. In otherembodiments, the oral dosage form is administered once daily at about600 mg/day 7 days per week. In other embodiments, the oral dosage formis administered once daily at about 700 mg/day 7 days per week. In otherembodiments, the oral dosage form is administered once daily at about800 mg/day 7 days per week.

In embodiments, the oral dosage form is administered at about 3-35mg/kg/day 5-7 days per week. In other embodiments, the oral dosage formis administered at about 3-35 mg/kg/day 5 days per week. In otherembodiments, the oral dosage form is administered at about 3-35mg/kg/day 6 days per week. In other embodiments, the oral dosage form isadministered at about 3-35 mg/kg/day 7 days per week. In otherembodiments, the oral dosage form is administered at about 6-12mg/kg/day 5-7 days per week. In other embodiments, the oral dosage formis administered at about 6-12 mg/kg/day 5 days per week. In otherembodiments, the oral dosage form is administered at about 6-12mg/kg/day 6 days per week. In other embodiments, the oral dosage form isadministered at about 6-12 mg/kg/day 7 days per week.

In some embodiments, the oral dosage form is a solid. In otherembodiments, the oral dosage form is a tablet. In other embodiments, theoral dosage form is a capsule. In other embodiments, the oral dosageform is immediate release. In other embodiments, the oral dosage form isextended release.

In embodiments, the oral dosage form is administered after the subjecthas fasted from food for at least about 8 hours. In other embodiments,the subject continues to fast from food for at least about 1 hour afteradministration. In other embodiments, the oral dosage form isadministered to the subject with food.

In embodiments, the compound of formula (I) is administrated as amonohydrate. In other embodiments, the compound of formula (I) isadministrated free of solvates, hydrates and salts.

In embodiments, the oral dosage form provides a T_(1/2) of about 3-20hours after a single administration. In other embodiments, the oraldosage form provides a T_(1/2) of about 5-10 hours after a singleadministration. In other embodiments, the oral dosage form provides aT_(1/2) of about 6-9 hours after a single administration. In otherembodiments, the oral dosage form provides a T_(1/2) of about 9-11 hoursafter a single administration. In other embodiments, the oral dosageform provides a T_(1/2) of about 6-7 hours after a singleadministration. In other embodiments, the oral dosage form provides aT_(1/2) of about 6 hours after a single administration. In otherembodiments, the oral dosage form provides a T_(1/2) of about 7 hoursafter a single administration.

In other embodiments, the oral dosage form provides a T_(1/2) of about 8hours after a single administration. In other embodiments, the oraldosage form provides a T_(1/2) of about 9 hours after a singleadministration. In other embodiments, the oral dosage form provides aT_(1/2) of about 10 hours after a single administration. In otherembodiments, the oral dosage form provides a T_(1/2) of about 11 hoursafter a single administration.

In embodiments, the oral dosage form provides a T_(max) of about 2-6hours after a single administration. In other embodiments, the oraldosage form provides a T_(max) of about 4-6 hours after a singleadministration. In other embodiments, the oral dosage form provides aT_(max) of about 2-4 hours after a single administration. In otherembodiments, the oral dosage form provides a T_(max) of about 2 hoursafter a single administration. In other embodiments, the oral dosageform provides a T_(max) of about 3 hours after a single administration.In other embodiments, the oral dosage form provides a T_(max) of about 4hours after a single administration. In other embodiments, the oraldosage form provides a T_(max) of about 5 hours after a singleadministration. In other embodiments, the oral dosage form provides aT_(max) of about 6 hours after a single administration.

In embodiments, the oral dosage form provides a C_(max) of about30-3,000 ng/mL after a single administration. In other embodiments, theoral dosage form provides a C_(max) of about 600-2,000 ng/mL after asingle administration. In other embodiments, the oral dosage formprovides a C_(max) of about 700-1,500 ng/mL after a singleadministration. In other embodiments, the oral dosage form provides aC_(max) of about 600-1,100 ng/mL after a single administration. In otherembodiments, the oral dosage form provides a C_(max) of about 700-1,100ng/mL after a single administration. In other embodiments, the oraldosage form provides a C_(max) of about 600-700 ng/mL after a singleadministration. In other embodiments, the oral dosage form provides aC_(max) of about 700-800 ng/mL after a single administration. In otherembodiments, the oral dosage form provides a C_(max) of about 800-900ng/mL after a single administration. In other embodiments, the oraldosage form provides a C_(max) of about 900-1,000 ng/mL after a singleadministration. In other embodiments, the oral dosage form provides aC_(max) of about 1,000-1,100 ng/mL after a single administration.

In embodiments, the oral dosage form provides an AUC_(0-t) (0-24 hours)of about 200-18,000 hr·ng/mL after a single administration. In otherembodiments, the oral dosage form provides an AUC_(0-t) (0-24 hours) ofabout 7,000-14,000 hr·ng/mL after a single administration.

In other embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 8,000-12,000 hr·ng/mL after a single administration. Inother embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 8,000-10,000 hr·ng/mL after a single administration. Inother embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 8,000-9,000 hr·ng/mL after a single administration. Inother embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 9,000-10,000 hr·ng/mL after a single administration. Inother embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 10,000-11,000 hr·ng/mL after a single administration. Inother embodiments, the oral dosage form provides an AUC_(0-t) (0-24hours) of about 11,000-12,000 hr·ng/mL after a single administration.

In embodiments, the tumor is ovarian cancer; metastatic breast cancer;adenocarcinoma of the pancreas; gastrointestinal cancer such ascolorectal adenocarcinoma or cancer of the esophagus, stomach, pancreas,small bowel, hepatobiliary tract, colon, rectum or anus; bladder cancersuch as metastatic bladder cancer, muscle invasive bladder cancer ornon-muscle invasive bladder cancer; cervical cancer; lung cancer;non-small cell lung cancer; or renal cell carcinoma. In otherembodiments, the tumor is pancreatic, bladder or colorectal cancer. Inother embodiments, the tumor is pancreatic cancer. In other embodiments,the cancer is bladder cancer. In other embodiments, the cancer iscolorectal cancer. In other embodiments, the cancer is colon cancer. Inother embodiments, the cancer is rectal cancer. In other embodiments,the tumor is non-small cell lung cancer and the compound of formula (I)or a hydrate, a solvate, or a pharmaceutically acceptable salt thereofis administered with cisplatin. In other embodiments, the tumor isresistant to gemcitabine. See Yang et al., Anticancer Research,34:6951-6960 (2014)(showing efficacy of RX-3117 in various xenograftmodels, even in tumors resistant to gemcitabine).

In embodiments, the subject is a mammal. In other embodiments, thesubject is a human.

Kits for Testing Efficacy of Treatment

Another aspect of the disclosure provides a kit for testing potentialefficacy of a compound of formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt in thetreatment of tumor, using an assay that measures one of protein kinases,p53, or UCK2 expression level in a sample of tumor cell.

In embodiments, the kit further comprises an assay that measures proteinCdc25C or p-Cdc25C expression level in a sample of tumor cell.

In any embodiment, the tumor cell is lung cancer cell. In otherembodiments, the tumor cell is non-small cell lung cancer cell. In otherembodiments, the tumor cell is pancreatic cancer cell or bladder cancercell.

Pharmaceutical Compositions

In any of the methods and kits provided herein, the compound of formula(I) may be in a pharmaceutical composition. Such pharmaceuticalcomposition can be prepared as any appropriate unit dosage form. Forexample, the pharmaceutical compositions can be formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, as drenches, tablets(such as those targeted for buccal, sublingual and systemic absorption,including over-encapsulation tablets), capsules (such as dry filled,hard gelatin, soft gelatin or over-encapsulation capsules), caplets,boluses, powders, sachets, granules, pastes, mouth sprays, troches,lozenges, pellets, syrups, suspensions, elixirs, liquids, liposomes,emulsions and microemulsions; or (2) parenteral administration by, forexample, subcutaneous, intramuscular, intravenous or epidural injectionas, for example, a sterile solution or suspension. Additionally, thepharmaceutical compositions can be formulated for immediate, sustained,extended, delayed or controlled release.

In one embodiment, the pharmaceutical composition is formulated for oraladministration. In embodiments, the pharmaceutical composition is intablet or capsule form. In other embodiments, the pharmaceuticalcomposition is in tablet form. In other embodiments, the pharmaceuticalcomposition is in capsule form. In other embodiments, the tablet orcapsule is formulated for immediate release. In other embodiments, thetablet or capsule is formulated for sustained, extended, delayed orcontrolled release.

Tablets can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared bycompressing in a suitable machine Compound (I) in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface-active or dispersing agent. Moldedtablets can be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletscan be optionally coated or scored and can be formulated so as toprovide sustained, extended, delayed or controlled release of Compound(I). Methods of formulating such sustained, extended, delayed orcontrolled release compositions are known in the art and disclosed inissued U.S. patents, including but not limited to U.S. Pat. Nos.4,369,174; 4,842,866; and the references cited therein. Coatings can beused for delivery of compounds to the intestine (see, e.g., U.S. Pat.Nos. 6,638,534; 5,217,720; 6,569,457; and the references cited therein).In addition to tablets, other dosage forms, such as capsules,granulations and gel-caps, can be formulated to provide sustained,extended, delayed or controlled release of Compound (I).

In embodiments, the pharmaceutical composition is formulated forparenteral administration. Examples of a pharmaceutical compositionsuitable for parenteral administration include aqueous sterile injectionsolutions and non-aqueous sterile injection solutions, each containing,for example, anti-oxidants, buffers, bacteriostats and/or solutes thatrender the formulation isotonic with the blood of the intendedrecipient; and aqueous sterile suspensions and non-aqueous sterilesuspensions, each containing, for example, suspending agents and/orthickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example, sealed ampules or vials, and can bestored in a freeze dried (lyophilized) condition requiring only theaddition of a sterile liquid carrier, such as water, immediately priorto use. In one embodiment, the pharmaceutical composition is formulatedfor intravenous administration.

In embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable excipient. A pharmaceutically acceptableexcipient may be any substance, not itself a therapeutic agent, used asa carrier, diluent, adjuvant, binder, and/or vehicle for delivery of atherapeutic agent to a patient, or added to a pharmaceutical compositionto improve its handling or storage properties or to permit or facilitateformation of a compound or pharmaceutical composition into a unit dosageform for administration. Pharmaceutically acceptable excipients areknown in the pharmaceutical arts and are disclosed, for example, inRemington: The Science and Practice of Pharmacy, 21st Ed. (LippincottWilliams & Wilkins, Baltimore, Md., 2005). As will be known to those inthe art, pharmaceutically acceptable excipients can provide a variety offunctions and can be described as wetting agents, buffering agents,suspending agents, lubricating agents, emulsifiers, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, andsweeteners. Examples of pharmaceutically acceptable excipients includewithout limitation: (1) sugars, such as lactose, glucose and sucrose;(2) starches, such as corn starch and potato starch; (3) cellulose andits derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, andhydroxypropylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin;(7) talc; (8) excipients, such as cocoa butter and suppository waxes;(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; and (22) other non-toxic compatible substances employedin pharmaceutical formulations.

In some embodiments, the pharmaceutical composition further comprises atleast one active agent in addition to RX-3117. The active agent may bean antineoplastic, chemotherapeutic, cytotoxic, radiotherapeutic(external-beam radiation therapy, internal radiation therapy, orsystemic radiation therapy) or any other agent capable of inducingapoptosis, sensitizing a cell to apoptosis, modulating protein kinase ortreating neoplasm, tumor or cancer. Examples of the active agentinclude: (1) antimetabolites, such as cytarabine, fludarabine,5-fluoro-2′-deoxyuiridine, gemcitabine, hydroxyurea or methotrexate; (2)DNA-fragmenting agents, such as bleomycin, (3) DNA-crosslinking agents,such as chlorambucil, cisplatin, cyclophosphamide and nitrogen mustard;(4) intercalating agents such as adriamycin (doxorubicin) andmitoxantrone; (5) protein synthesis inhibitors, such as L-asparaginase,cycloheximide, puromycin and diphtheria toxin; (6) topoisomerase Ipoisons, such as camptothecin and topotecan; (7) topoisomerase IIpoisons, such as etoposide (VP-16) and teniposide; (8)microtubule-directed agents, such as colcemid, colchicine, paclitaxel,vinblastine and vincristine; (9) kinase inhibitors such as flavopiridol,staurosporin and 7-hydroxystaurosporine; (10) polyphenols such asquercetin, resveratrol, piceatannol, epigallocatechine gallate,theaflavins, flavanols, procyanidins, betulinic acid and derivativesthereof; (11) hormones such as glucocorticoids and fenretinide; (12)hormone antagonists, such as tamoxifen, finasteride and LHRHantagonists; and (13) death receptor agonists, such as tumor necrosisfactor a (TNF-α), tumor necrosis factor 3 (TNF-β), LT-β (lymphotoxin-β),TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1) ligand, TRAMP (DR3, Apo-3)ligand, DR6 ligand and fragments and derivatives thereof.

In embodiments, the amount of the compound of formula (I) or hydrate,solvate, or pharmaceutically acceptable salt in the pharmaceuticalcomposition is between about 0.1% and about 100% by weight. In otherembodiments, the amount is between about 0.5% and about 99.5% by weight.In embodiments, the amount is between about 10% and about 95% by weight.In embodiments, the amount is between about 15% and about 90% by weight.In embodiments, the amount is between about 80% and about 90% by weight.In embodiments, the amount is between about 80% and about 85% by weight.In embodiments, the amount is at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, or at least 80%. In embodiments, thepharmaceutical composition is an oral dosage form. In embodiment, thepharmaceutical composition is a tablet.

Methods of Administration

In any of the methods provided herein, administration of the compound orpharmaceutical composition may be via any accepted mode known in theart, such as orally or parenterally. The term “parenterally” includeswithout limitation subcutaneously, intravenously, intramuscularly,intraperitoneally, intravesically, intrathecally, intraventricularly,intrasternally, intracranially, by intraosseous injection and byinfusion techniques. In one embodiment, the compound or pharmaceuticalcomposition is administered orally. In other embodiments, the compoundor pharmaceutical composition is administered parenterally. In otherembodiments, the compound or pharmaceutical composition is administeredintravenously.

In one embodiment, the compound or pharmaceutical composition isadministered orally at a dose or dosage as disclosed herein, such as inMethods of Treating or Preventing Tumor, supra. In any of the methodsdisclosed herein, the compound or pharmaceutical composition may beadministered based on a weight based dose. In other embodiments, theeffective amount is about 0.01 to about 100 mg/kg/day or about 3 toabout 35 mg/kg/day. In embodiments, the effective amount is about 6 to12 mg/kg/day.

The dose level can be adjusted for intravenous administration. In suchcase, the compound or pharmaceutical composition can be administered inan amount of between about 0.01 μg/kg/min to about 100 μg/kg/min.

Combination Therapy

In any of the methods of treating or preventing a tumor provided herein,the method may further comprise administering RX-3117 with one or moreadditional anti-tumor agent or radiation to the subject. In oneembodiment, the method further comprises administering radiation to thesubject. In other embodiments, the method further comprisesadministering one or more additional anti-tumor agent to the subject.

The additional anti-tumor agent or radiation may be administered before,after, or during administration of the compound of formula (I) orhydrate, solvate, or pharmaceutically acceptable salt thereof. In oneembodiment, the additional anti-tumor agent or radiation is administeredbefore administration of the compound of formula (I) or hydrate,solvate, or pharmaceutically acceptable salt thereof. In otherembodiments, the additional anti-tumor agent or radiation isadministered after administration of the compound of formula (I) orhydrate, solvate, or pharmaceutically acceptable salt thereof. In otherembodiments, the additional anti-tumor agent or radiation isadministered during administration of the compound of formula (I) orhydrate, solvate, or pharmaceutically acceptable salt thereof. In otherembodiments, the additional anti-tumor agent and the compound of formula(I) or hydrate, solvate, or pharmaceutically acceptable salt thereof areformulated into a pharmaceutical composition for concurrentadministration.

Radiosensitizing Effect

The radiosensitizing effect of RX-3117 was studied in A2780 ovariancancer cells and NSCLC cell lines. RX-3117 was found to have a scheduledependent radiosensitizing effect, but only at preincubation (dosemodifying factors: 1.4-1.8), observed at pulse and fractionatedirradiation. Radiosensitizion was also seen in a 3-dimensional spheroidmodel. At a low radiosensitizing concentration, RX-3117 in combinationwith radiation led to an accumulation of cells in S-phase, which wasaccompanied by an increase of all cell cycle proteins such as p-Chk2 andp-cdc25C. In addition, RX-3117 caused cell killing due to DNA damage. Inconclusion, the in vitro experiments showed radiosensitizing effect ofRX-3117.

Lung cancer patients are standardly being treated with surgery and thosein advanced disease receive chemotherapy (Baas P, Belderbos J S A, SenanS, Kwa H B, van Bochove A, van Tinteren H, Burgers J A and van MeerbeeckJ P: Concurrent chemotherapy (carboplatin, paclitaxel, etoposide) andinvolved-field radiotherapy in limited stage small cell lung cancer: aDutch multicenter phase II study. Br J Cancer 94: 625-30, 2006). Acombination of the cytidine analog, gemcitabine and cisplatin is beingused in the clinic to treat the disease (El-Naggar M, Ebbing E,Bijnsdorp I, van den Berg J and Peters G J: Radiosensitization bythymidine phosphorylase inhibitor in thymidine phosphorylase negativeand overexpressing bladder cancer cell lines. Nucleosides NucleotidesNucleic Acids 33: 413-21, 2014). However, resistance is a limitingfactor, and, therefore, there is a need for novel drugs which bypass theresistance mechanism and ideally show effective combination properties.

Mechanism of RX-3117 Action

RX-3117 is an analog of cytidine (FIG. 14). It has a modification on theribose molecule consisting of a carbon-fluorine bond instead of oxygenand a double bond (Choi W J, Chung H-J, Chandra G, Alexander V, Zhao LX, Lee H W, Nayak A, Majik M S, Kim H O, Kim J-H, Lee Y B, Ahn C H, LeeS K and Jeong L S: Fluorocyclopentenyl-cytosine with broad spectrum andpotent antitumor activity. J Med Chem 55: 4521-5, 2012). As shown inFIG. 21, RX-3117 enters the cell via human equilibrative nucleosidetransporter (hENT) (Peters G J, Smid K, Vecchi L, Kathmann I, SarkisjanD, Honeywell R J, Losekoot N, Ohne O, Orbach A, Blaugrund E, Jeong L S,Lee Y B, Ahn C-H and Kim D J: Metabolism, mechanism of action andsensitivity profile of fluorocyclopentenylcytosine (RX-3117). Invest NewDrugs 31: 1444-57, 2013). In the cell RX-3117 is phosphorylated byuridine/cytidine kinase 2 (UCK2) to its monophosphate form, i.e.,RX-3117 is activated by UCK2 to RX-3117 MP. RX-3117 tri-phosphate(RX-3117 TP) is incorporated into the RNA. Its RX-3117 di-phosphate(RX-3117 DP) is reduced to deoxy-di-phosphate (dRX-3117 DP) byribonucleotide reductase (RR) before the incorporation into the DNA.RX-3117 is a poor substrate for cytidine deaminase (CDA) (id.). Potenttumor growth inhibition by RX-3117 in gemcitabine resistant mouse modelswas recently demonstrated (Yang M Y, Lee Y B, Ahn C-H, Kaye J, Fine T,Kashi R, Ohne O, Smid K, Peters G J and Kim D J: A novel cytidineanalog, RX-3117, shows potent efficacy in xenograft models, even intumors that are resistant to gemcitabine. Anticancer Res 34: 6951-9,2014). A radiosensitizing effect of RX-3117 has been investigated andresults shown below.

RX-3117 is categorized as a pyrimidine analog (Peters G J: Noveldevelopments in the use of antimetabolites. Nucleosides NucleotidesNucleic Acids 33: 358-74, 2014), similar to other analogs such asgemcitabine and azacytidine, which are extensively being used in theclinic. Gemcitabine is a potent radiosensitizer, which increasesionization induced DNA damage repair (Morgan M a, Parsels L a, Maybaum Jand Lawrence T S: Improving gemcitabine-mediated radiosensitizationusing molecularly targeted therapy: a review. Clin Cancer Res 14:6744-50, 2008).

In addition, the cytidine analogs 5-azacytidine (aza-C, Vidaza™) and5-aza-2′-deoxy-cytidine (decitabine, Dacogen®) are being used in theclinic for treatment of myelodysplastic syndrome (MDS) (Peters G J:Novel developments in the use of antimetabolites. NucleosidesNucleotides Nucleic Acids 33: 358-74, 2014). Two main mechanisms ofanti-tumor effect of these drugs are DNA methyltransferase (DNMT)inhibition and cytotoxic incorporation in RNA and/or DNA (Kaminskas E,Farrell A, Abraham S, Baird A, Hsieh L-S, Lee S-L, Leighton J K, PatelH, Rahman A, Sridhara R, Wang Y-C and Pazdur R: Approval summary:azacitidine for treatment of myelodysplastic syndrome subtypes. ClinCancer Res 11: 3604-8, 2005). After uptake into the cells, aza-C isphosphorylated to 5-azacytidine monophosphate (aza-CMP) by UCK2 and toaza-CDP and aza-CTP by pyrimidine nucleotide kinases. However, aza-C isinactivated by deamination by CDA. RR reduces aza-CDP to aza-dCDP, whichis phosphorylated by nucleoside diphosphate kinase to aza-dCTP. Aza-dCTPis then incorporated into DNA, resulting in DNA synthesis inhibition(Veselý J: Mode of action and effects of 5-azacytidine and of itsderivatives in eukaryotic cells. Pharmacol Ther 28: 227-35, 1985).Stoichiometric binding of aza-dCTP with DNMT will result in DNAhypomethylation (Jones P A: Effects of 5-azacytidine and its2′-deoxyderivative on cell differentiation and DNA methylation.Pharmacol Ther 28: 17-27, 1985). Aza-dCTP can also be formed from5-aza-2′-deoxycytidine by direct phosphorylation catalyzed bydeoxycytidine kinase (dCK) and nucleotide kinases. DNA hypermethylationat CpG islands has been described in different malignancies includingMDS (Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh L-S, Lee S-L,Leighton J K, Patel H, Rahman A, Sridhara R, Wang Y-C and Pazdur R:Approval summary: azacitidine for treatment of myelodysplastic syndromesubtypes. Clin Cancer Res 11: 3604-8, 2005). On the other hand, aza-CTPincorporates into RNA disrupting metabolism of cytoplasmic and nuclearRNA protein synthesis (Glover A B and Leyland-Jones B: Biochemistry ofazacitidine: a review. Cancer Treat Rep 71: 959-64, 1987). One mechanismof resistance to azacytidine is a point mutation in the UCK2 gene, whichresults in an inactive metabolite (Sripayap P, Nagai T, Uesawa M,Kobayashi H, Tsukahara T, Ohmine K, Muroi K and Ozawa K: Mechanisms ofresistance to azacitidine in human leukemia cell lines. Exp Hematol 42:294-306.e2, 2014). The mechanism underlying resistance to aza-dCTP is adeficiency of dCK (Peters G J: Novel developments in the use ofantimetabolites. Nucleosides Nucleotides Nucleic Acids 33: 358-74,2014).

As shown in FIG. 22, RX-3117 can also down-regulate DNMT1(Peters G J,Smid K, Vecchi L, Kathmann I, Sarkisjan D, Honeywell R J, Losekoot N,Ohne O, Orbach A, Blaugrund E, Jeong L S, Lee Y B, Ahn C-H and Kim D J:Metabolism, mechanism of action and sensitivity profile offluorocyclopentenylcytosine (RX-3117). Invest New Drugs 31: 1444-57,2013), but seems to act differently than aza-C. Furthermore, severalcytidine analogs, but not all, show a radiosensitizing effect.Therefore, the potential radiosensitizing effects as well as thepotential mechanisms, such as cell cycle effects and cell killing, ofRX-3117 were evaluated. RX-3117 was accordingly shown to have aradiosensitizing effect.

Pre-incubation with RX-3117 had the best radiosensitizing effect and 4of the 5 cell lines tested were sensitized by RX-3117. The gemcitabineresistant SW1573/G- was sensitized by RX-3117 with almost the sameefficacy as its wild type. RX-3117 also showed a radiosensitizing effectin two spheroid models.

Nucleoside analogs have been shown to enhance irradiation induced cellkill (Shewach D S and Lawrence T S: Antimetabolite radiosensitizers. JClin Oncol 25: 4043-50, 2007). The radiosensitizing effect is thought tobe carried out by targeting deoxyribonucleotide biosynthesis (which areneeded for DNA replication) or DNA polymerases (Shewach D S and LawrenceT S: Antimetabolite radiosensitizers. J Clin Oncol 25: 4043-50, 2007;Lawrence T S, Blackstock A W and McGinn C: The mechanism of action ofradiosensitization of conventional chemotherapeutic agents. Semin RadiatOncol 13: 13-21, 2003). An example of deoxynucleotide pool deregulationis the TS inhibitor 5-fluoro-2′-deoxyuridine (FdUrd). TS inhibitorscause deoxynucleotide pools imbalance resulting in DNA synthesisinhibition and S phase arrest (Hwang H S, Davis T W, Houghton J a andKinsella T J: Radiosensitivity of thymidylate synthase-deficient humantumor cells is affected by progression through the G1 restriction pointinto S-phase: implications for fluoropyrimidine radiosensitization.Cancer Res 60: 92-100, 2000). Imbalance in deoxynucleotide pools causesincorporation of incorrect nucleotides (Ingraham H A, Tseng B Y andGoulian M: Nucleotide levels and incorporation of 5-fluorouracil anduracil into DNA of cells treated with 5-fluorodeoxyuridine. MolPharmacol 21: 211-6, 1982). The concentration that is needed forradiosensitizing effect to be achieved is not necessarily theconcentration which is needed for cytotoxic effect. Lower concentrationsof drugs can establish the radiosensitization (Hwang H S, Davis T W,Houghton J a and Kinsella T J: Radiosensitivity of thymidylatesynthase-deficient human tumor cells is affected by progression throughthe G1 restriction point into S-phase: implications for fluoropyrimidineradiosensitization. Cancer Res 60: 92-100, 2000). A low dose of RX-3117induced radiosensitizing effect in the clonogenic assay, in the3-dimensional model and fractionated irradiation schedule. Also, thedouble strand DNA breaks induced by RX-3117 were dose dependent.

RX-3117 has been shown to be a potent schedule dependent radiosensitizerin four out of five cell lines, with potential for clinical applicationwhere combination treatment is considered in NSCLC. This combinationtreatment may apply to other tumor types (such as prostate, skin, headand neck, throat, larynx, breast, brain, colorectal, bone, leukemia,ovarian, and uterine cancer.)

Improvements of the Process for Making RX-3117

U.S. Pat. No. 7,405,214 discloses an 11-step synthesis of RX-3117 fromD-ribose. The synthesis uses an expensive catalyst which poses achallenge for implementation in large scale plant production. U.S. Pat.No. 9,150,520 improved the synthesis disclosing a shorter route for thepreparation of RX-3117 through(3R,4R,6aR)-tert-butyl-(5-fluoro-2,2-dimethyl-6-trityloxymethyl-4,6a-dihy-dro-3aH-cyclopenta[1,3]dioxol-4-yloxy)-diphenyl-silane(ASM11) to 4-amino-1-(3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-d-ihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14). However, the prior synthesis of ASM11 to INT14 required theintermediates to be isolated in each step. Thus, the process poses costand time constraints, particularly if scaled up for commercialmanufacturing.

The present invention provides an improved process of preparing RX-3117,which is commercially viable for large scale production. The processtelescope the synthesis of ASM11 to INT14 without the requirement ofisolating each of the intermediate materials, thereby reducing costwhile improving efficiency. More specifically, the current inventionprovides a continuous process with three stages to telescope thesynthesis of ASM11 to INT14. The present invention also provides aprocess to afford RX-3117 monohydrate (RX-3117-MH) in fixed vessels tosignificantly reduce the cost of manufacture. By telescoping three stepsinto a single step, the present process removes the requirement toconcentrate an intermediate to a residue. These improvements are basedon unexpected benefits when substituting reagents that are not readilyapparent to person skilled in the art.

Furthermore, the present invention provides optimized reaction andisolation conditions to increase the nitrogen to oxygen (N/O)selectivity at Stage 3, where cytosine is added to(3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate (INT13) to make INT14. In the improved process, theratio of the N- to O-isomers was improved to 99.03:0.97 from thepreviously optimized value of 88:12. The fixed vessel manufacturingprocess of the present invention achieves the cost benefits of operationof a scaled-up manufacturing of the desired product in monohydrate form.

Scheme 1 below illustrates an improved process for preparing RX-3117MH.

Stage 1—Process Improvements for Deprotection of ASM11 to Form INT12

In Stage 1 of the process, 2-methyl-tetrahydrofuran was used as theprocess solvent. This modification allows a work-up to be performedwithout the need to concentrate the intermediate (3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol(INT12) and avoid the solvent exchange of the intermediate into methyltert-butyl ether (MTBE), which was used in the prior process.

In addition, the reaction in-process control (IPC) was changed fromusing TLC to quantitative ¹H NMR method. The process was furtheroptimized by using azeotropic removal of water instead of chemicaldrying. The use of 2-methyl-tetrahydrofuran as the process solventallowed INT12 in solution to be used directly in Stage 2 of the processwithout further isolation or purification.

Stage 2—Process Improvements for Mesylation of INT12 to Form INT13

The solution of INT12 in 2-methyl-tetrahydrofuran was telescopeddirectly into Stage 2 to prepare(3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate (INT13). This improved process eliminated the use ofthe environmentally undesirable dichloromethane as the reaction solvent.Furthermore, the process used an ammonium chloride wash to furthercontrol residual triethyl amine. The work up volumes were reduced to amaximum process volume at 12.5 vol., a reduction from 14 volumes. Again,the process was further optimized by using azeotropic removal of waterinstead of chemical drying. The use of 2-methyl-tetrahydrofuran as theprocess solvent allowed INT13 in solution to be used directly in Stage 3of the process.

Stage 3—Process Improvements for the Addition of Cytosine to INT13 toForm INT14

The solution of INT13 in 2-methyl-tetrahydrofuran was telescopeddirectly into Stage 3 to make INT14. Dimethyl sulfoxide (DMSO) wasretained as the reaction solvent and the removal of2-methyl-tetrahydrofuran was performed by distillation. A specificationof 27% w/w of 2-methyl-tetrahydrofuran versus product was found to allowthe stage 3 reaction to perform well. The inventors conducted ascreening of bases (inorganic and amine) and found that cesium carbonateoffered the highest chemo-selectivity and most rapid reaction rate. Theinventors also conducted a screening of reaction solvents and found DMSOto be the most suitable solvent for the reaction.

Furthermore, the inventors studied the impact of reagent charges,temperature and concentration on the selectivity of the N- vs O-isomersof INT14. In accordance with the improved process of the presentinvention, the ratio of the N- to O-isomers was improved to 99.03:0.97using solvent extraction and recrystallization/precipitation from thepreviously optimized 88:12, which was isolated using SiO₂ columnchromatography. The inventive process eliminates the need of columnchromatography and also provides the desired N-isomers at over 99%. Inparticular, the inventors found that chemoselectivity was effectedprimarily by the reaction temperature. In particular, lowering thereaction temperature slowed the rate of conversion to product. Thereaction condition was further improved by increasing the charging ofbase and cytosine from 2.0 equivalents to 2.5 equivalents. The reactiontemperature was reduced from 40° C. to 35° C. In addition, the work upprocedure was modified to reduce the total process volume from 22 vol to12.5 vol to improve throughput. The work up solvent was changed fromethyl acetate to isopropyl acetate to allow the reaction mixture to movedirectly into isolation without the requirement to exchange solvent. Thework up procedure was also modified to start with the tautomerization asit was found that INT14 was more soluble following the acetic acidtreatment. The isolation of INT14 was modified to initially precipitatethe product from isopropyl acetate at high volume prior to reducing thevolume and adding n-heptane. This improvement was found to preventoiling and adherence to the vessel prior to isolation. Thus, an overallimproved synthesis with increased selectivity was achieved bysimultaneously changing multiple reaction parameters. The modificationof temperature and concentration were found to have a positive impact onrate of conversion and chemoselectivity of the N/O alkylation.

Stage 4—Process for Deprotecting INT14 to Form RX-3117 Anhydrous

The original conditions of 2 μM HCl in ethanol were found to be the moststable for the product and were retained. However, the process wasimproved by reducing the reaction temperature from 60° C. to 50° C. toaid solubility. The trityl alcohol by-product was removed using methyltert-butyl ether (MTBE) washes. The product in the aqueous phase wastelescoped directly into the Stage 5 isolation following the resin saltrelease.

Stage 5—Process for Isolating RX-3117 Monohydrate

The solution of RX-3117MH was telescoped directly into Stage 5. Thecombined Stage 4 and Stage 5 with minor modifications to the procedureoptimized yield and operability on scale. The RX-3117-MH was dried on afilter under air, which controlled acetonitrile quantities to below theICH guideline while retaining the water content. This improved processremoved the time-demanding requirement to first dry and then re-hydratethe product to afford a crystalline product. The isolated product usingthe improved process has a purity of (99.83%), which was comparable inpurity with the custom synthesis of the product in small quantities.

Other Improvements of the Process for Making Starting Materials ofRX-3117

Other process improvements for the synthesis of the starting materialsof RX-3117 are possible. Synthesis of ASM11 using differentintermediates and protecting groups are shown below in Schemes 2 and 3,respectively.

Synthesis of ASM11 by Bromo Intermediates

In U.S. Pat. No. 9,150,520, iodoform was used in step 3 of the reactionto convert RXN-2 to RXN-3. In the present invention, the use ofbromoform or a mixed bromo-iodomethane to affords bromointermediatesinstead of iodointermediates. The bormointermediates can be more stablethan their iodo derivate. Therefore, the overall yield and purity canincrease as a result.

Synthesis of ASM11Bn by Benzyl Protection of 5-Hydroxyl Group

Unexpectedly, changes in protecting groups placed in early intermediatescan have dramatic effects on reaction steps conducted later in theprocess, without requiring modification of numerous steps along the way.For example, changing the trityl protecting group to benzyl in Step 2could improve the yield on the fluorination in Step 10 of the reaction.These improvements can be made without further modification of theoverall procedure.

Synthesis of ASM11 by Ring Closing Metathesis

The inventors of the present invention also developed schemes for thesynthesis of ASM11 by the employment of ring closing metathesis. InScheme 4, a ring closing metathesis reaction is used to form the5-member ring moiety. Ruthenium of the Grubb's catalyst is recoverable,further improving scale up processes by reducing waste and cost.

Synthesis of Intermediate RXN-6 by Ring Closing Metathesis

The synthesis of intermediate RXN-6 can be accomplished by ring closingmetathesis, including RXN-5 and to introduce the fluorine atom to thefive member ring by making a fluorinated RXN-6. As shown in Scheme 5, aring closing metathesis reaction is used to form the 5-member ringmoiety (Fluoro-RXN-6). As in Scheme 4, the ruthenium of the Grubb'scatalyst is recoverable.

Synthesis of Intermediate RXN-6 by Nucleophilic Fluorination Via anEpoxide

The synthesis of intermediate RXN-6 by an alternative nucleophilicfluorination via an epoxide can be provided. Scheme 6 shows theformation of an epoxide ring from the starting material(3aR,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-one.The epoxide is opened by nucleophilic fluorination using, for example,potassium fluoride. As an alternative, other fluoride source, forexample, tetrabutylammonium fluoride, can also be used to open theepoxide ring. The elimination of water is a difficult step in thisprocess and alternative dehydrating agents, such as, for example,carbomethoxy sulfamoyl triethylammonium salts, can be used to arrive atthe modified intermediate Flouro-RXN6-TBDPS.

Synthesis of Intermediate RXN6 by Aldol Condensation

The synthesis of intermediate RXN6 can also be accomplished using AldolCondensation to introduce a fluorine atom to the five member ring bymaking a fluorinated RXN-6. As shown in scheme 7, the fluorine atom isintroduced early on to form the fluorinated derivative of RXN6(Fluoro-RXN6). An internal Aldol Condensation can form the 5-memberedring with the vinyl fluorine moiety in place.

Alternate Synthesis of Intermediate Fluoro-RXN6

Additional alternatives for the synthesis of intermediate Fluoro-RXN-6are shown in Schemes 8 and 9. In Scheme 8, a shorter route is obtainedby reacting D-ribolactone 1 with phosphonate 2 to generate intermediate3. The inventors found that the D-ribolactone derivative 1 does notreact readily with dimethyl fluoroalkylphosphonate, but a betterreactivity can be obtained using dimethyl carbalkoxymethylphosphonate.The intermediate 3 then undergoes a Hundsdiecker iododecarboxylation toform intermediate 4, which can then undergo nucleophilic substitutionusing tetra-n-butylammonium fluoride (TBAF).

Scheme 9 (below) provides an even shorter route by reactingD-ribolactone 1 with an alkyl phenyl sulfone 7, which is prepared viaelectrophilic fluorinated-reagents, to form intermediate 8. TheD-ribolactone derivative reacts more readily with lithio fluoroalkylsulfone 7 than with fluoroalkylphosphonates. The intermediate 8 can beconverted to intermediate 9, which will undergo elimination to formF-RXN6. Alternatively, a more efficient but more expensive option is touse a fluorinated-tetrazolyl sulfone 10 in place of lithio fluoroalkylsulfone 7.

Synthesis Using Different Chiral Sources

The present synthesis can also be achieved by using different chiralsources as starting materials.

In Scheme 10 (below),(2S,3S,4R,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol isused as an alternative chiral starting material to generate ASM-11.

EXAMPLES

The following examples are presented for illustrative purposes andshould not serve to limit the scope of the disclosed subject matter.

IC₅₀ values for cell-lines A549, SW1573, and SW1573/G used herein are8.3 μM, 13.7 μM and 7.3 μM, respectively, as reported in Peters et al.,“Metabolism, mechanism of action and sensitivity profile offluorocyclopentenylcytosine (RX-3117),” Investigational New Drugs,December 2013, Vol. 31, No. 6, pp. 1444-1457 (available online athttp://link.springer.com/article/10.1007/s10637-013-0025-x).

Example 1: Effect of RX-3117 on Non-Small Cell Lung Cancer Cell Lines

The effect of RX-3117 on cell cycle regulation and cell death in humannon-small cell lung cancer (NSCLC) cell lines A549 (adenocarcinoma),SW1573 (alveolar carcinoma) and SW1573/G- (SW1573 cell line resistant togemcitabine) and H460 (large cell carcinoma) was assessed. The A549 andH460 cell lines were obtained from American Type Culture Collection(Manassas, Va., USA). The SW1573 cell line, obtained from Dr. Johan vanRijn (see Keiser et al., Cancer Research, 49:2988-2993 (1989)), alsoserved as the parental cell line for SW1573/G-. A549, SW1573, andSW1573/G- and H460 were kept in exponential growth in T25 flasks(Greiner Bio-One GmbH, Frickenhausen, Germany) and cultured inDulbecco's minimal essential medium (DMEM) or RPMI 1640 (A549)supplemented with 10% heat-inactivated fetal bovine serum (FBS) 1%streptomycin and penicillin 20 mM HEPES and maintained in 37° C. watersaturated atmospheres of 5% CO₂. For the cell cycle distribution,1.5×10⁵ cells were seeded in a T25 flask (Greiner Bio-One GmbH) andcultured for 72 hours after which 1 μM of RX-3117 was added andincubated for 24 hours. The cells were then harvested. After thetreatment, the medium was collected in a 15 ml tube (Greiner Bio-OneGmbH). The cells were washed with ice cold PBS and trypsinized (Lonza)at 37° C. The collected medium was used to inactivate trypsin of thecorresponding sample and again collected in the 15 ml tube. The cellswere centrifuged with the standard centrifuge program, 5 minutes at 4°C. and 12,000 rpm. The medium was removed, pellet washed with 1 mlPBS/0.01% BSA and centrifuged. After removal of the supernatant, thecells were fixed with 1 ml 70% ethanol and incubated at −20° C. for atleast 24 hours. Subsequently, the cells were centrifuged, washed with 1ml PBS/0.1% BSA and transferred to a FALCON FACS tube (BD, FranklinLakes, N.J., USA). The cells were centrifuged, and the supernatant wasremoved followed by the addition of 0.5 μg propidium iodide (PI) (Sigma,St. Louis, Mo., USA), 0.1% trisodium citrate (Riedel-de Haen,Sigma-Aldrich Laborchemikalien GmbH, St. Louis, Mo., USA), 0.1% TritonX-100™ (Merck) and 0.1 mg/ml RNase (Sigma) (PI solution) to the samples.Subsequently, for at least 15 minutes, the cells were incubated on icewith the PI solution to stain the DNA before starting the analysis. Thecells stained with the PI solution were analyzed by FACSCalibur™ (BDBiosciences, Mount View, Calif., USA). Data was analyzed with CellQuest™Pro software.

The mechanism of cell cycle arrest was investigated by measuring cellcycle proteins expression using western blotting. The influence ofRX-3117 on protein expression during different treatment conditions wasanalyzed by western blot. Cells were lysed using cell lysis buffer 1×(Cell Signaling, Danvers, Mass., USA) containing 4% protease inhibitorcocktail (Roche Diagnostics, Mannheim, Germany) on ice for 30 minutesand centrifuged for 10 minutes at 4° C. at 14,000 rpm. The proteincontaining supernatant was collected and the Bio-Rad assay was performedto determine protein amount as described in Lemos et al.,Pharmacogenomics, 12(2):159-70 (2011). The following antibodies wereused for protein expression: DNMT1 (Cell Signaling, 1:1000 #5032S),DNMT3A (Cell Signaling 1:1000 #2160S), DNMT3B (Abcam, 1; 1000), Chk2(Cell Signaling 1:1000 #6334P), Chk1 (Cell Signaling 1:1000), p-CDC25C(Cell Signaling 1: 1000 #4901S), Cdk1 (Cell Signaling 1:1000 #9112S),Cdk2 (Cell Signaling 1:1000 #2546S), wee1 (Cell Signaling 1:1000),S139-γH2A.X (Cell Signaling, 1:1000), β actin (Sigma, 1: 10,000),Caspase 9 (Cell Signaling, 1:1000), PARP (Roche 2003, 1:1000), p53 (CellSignaling, 1:1000, #9282). The antibodies were diluted in 1:1 solutionRockland buffer (Rockland Inc., Philadelphia, Pa., USA) and PBSsupplemented with 0.05% Tween® 20. The proteins were separated in 20%SDS-PAGE and transferred to a PVDF membrane. For fluorescent signalsecondary antibodies goat anti-mouse InfraRedDye and goat anti-rabbitInfraRedDye were used. The proteins were detected by an Odyssey InfraRedImager (Li-COR Bioscience, Lincoln, Nebr., USA).

Abbreviations used herein denote the following:

BSA=bovine serum albumin

HEPES=2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

PBS=phosphate buffered saline

PVDF=polyvinylidene difluoride

RPM=revolutions per minute

SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis

Cell Cycle

At a dose of 1 μM, RX-3117 induced accumulation of A549, SW1573,SW1573/G- and H460 cells in the G1 phase after 24 hour exposure (FIG.1). At a higher dose of 5×IC₅₀, RX-3117 induced the accumulation ofA549, SW1573 and SW1573/G- cells in the S-phase (FIG. 2).

Caspase Activation

RX-3117 decreased pro-caspase 9 in SW1573 cells and A549 cells after 24hour exposure to increasing concentrations of RX-3117. Reduction ofpro-caspase 9 indicates activation of caspase and subsequentialapoptosis induction (FIGS. 3 and 4).

DNMT Protein

RX-3117 down regulates maintenance of DNA methyltransferase 1 (DNMT1) inA549 cells (FIG. 23) at higher dose and increased DNMT3A and DNMT3Bexpression levels in A549 cells. A proposed mechanism for this downregulation is shown in FIG. 22.

DNA Damage

RX-3117 induced double-strand breaks (DSB) as indicated by biomarkerγH2A.X (phospho S139) in SW1573 cells after 48 hour exposure (FIG. 5).RX-3117 induced cleaved PARP after 24 hour exposure to increasingconcentrations of RX-3117 (FIG. 6). Cleaved PARP indicates activatedcaspases activity in apoptotic cells. At 1 μM and 5 μM, RX-3117increased p53 expression levels in A549 cells (FIG. 7). At 10 μM,RX-3117 increased Chk1 and Cdk2 expression levels, while decreasingp-Cdc25C expression levels in SW1573 cells after 48 hour exposure (FIG.8). DNA damage is induced by RX-3117 triggers the Chk1 pathway. TheATR/Chk1 pathway is induced by DNA replication stress and DSB. RX-3117decreased wee1 expression levels in SW1573 cells after 24 hour exposureto increasing concentrations of RX-3117 (FIG. 9). FIG. 24 is a diagramshowing the potential effects on cell cycle proteins and regulation ofthe cell cycle by checkpoint kinases Chk1 and Chk2 after damageinduction, RX-3117 may have activity along several of these pathways.

Apoptosis Induction

At a dose of 5×IC₅₀, RX-3117 induced apoptosis in PI stained A549 andSW1573 cells in the sub-GI phase after 24 and 48 hour exposure (FIG.10). At 5 μM (for A549) and 10 μM (for SW1573), RX-3117 inducedapoptosis in Annexin V stained A549 and SW1573 cells in the sub-GI phaseafter 24, 48, 72 and 96 hour exposure (FIG. 11).

Results

The results suggest that cell cycle arrest was time, concentration andcell line dependent. In A549, H460 and SW1573 cells, 24 hour exposure to1 μM RX-3117 increased the accumulation of cells in the G1 phase (about20-40%) and in the S-phase (to a lesser extent), but decreased theaccumulation of cells in the G2/M phase. Thus, low dose of RX-3117induces G1 accumulation and high dose of RX-3117 induces S phaseaccumulation. No cell kill was observed at 24 hour exposure, but cellkill was observed at 48 hour exposure (15% in SW1573 and 8% in A549cells) accompanied by γH2AX induction. In A549 cells, the effect ofRX-3117 on the cell cycle distribution was most pronounced at 48 hourexposure with 45% accumulation in S phase. S-phase accumulation is timedependent. Treatment with RX3117 increased p53, Chk1, Chk2 and Cdk2expression levels, but decreased Cdc25C and p-Cdc25C expression levels.RX-3117 increased wee1 expression levels mostly after 48 hours. RX-3117appeared to induce apoptosis through SSB and DSB. Cleaved PARP in SW1573cells indicates upregulated caspase activity in apoptotic cells.Reduction of pro-caspase 9 in A549 cells indicates activation of caspaseand subsequential apoptosis induction. In conclusion, DNA damage inducedby RX-3117 triggered apoptosis on one hand and increased Chk1 and Chk2expression levels on the other hand. Without being limited to anymechanism of action, it is believed that the phosphorylated Chk1 andChk2 may have triggered phosphorylation of Cdc25C and provoked itsdegradation, which resulted in decreased Cdk1 levels and thusaccumulated cells in S-phase.

Example 2: Efficacy of RX-3117 in Syngeneic MC38 Murine Colon CancerXenograft Model

Following the protocol described below, the effect of RX-3117 on tumorgrowth in a syngeneic model using female C57BL/6 mice with MC38 murinecolon cancer was examined. Tumor growth was measured in a treatmentgroup compared to a control (vehicle treated) group (see Table 1 belowfor dosing scheme and treatment regimen). The results of this study(Table 2) demonstrate that the addition of RX-3117 to a programmed deathreceptor 1 (PD-1) inhibitor, RMP1-14, had an additive effect in theinhibition of tumor growth (80% RX-3117 alone, 93% RMP1-14 alone, versus99% in combination of two agents). Combination of the two agents alsoresulted in higher number of mice (9 mice) with partial regression andcomplete regression with 7 animals showing tumor free survival, comparedto 4 animals with partial regression and complete regression in theRMP1-14 alone group with 2 showing tumor free survival. All results wereobtained without any adverse effects to the mice in the combinationgroup.

Briefly, the method is described as follows. The cells were harvestedduring exponential growth and re-suspended with phosphate bufferedsaline. Each test animal received a subcutaneous (s.c.) injection of1×10⁶ tumor cells into the right flank and tumor growth was monitored asthe average tumor size approaches the target range of 60-100 mm³.Dosing, based on Table 1 started as each animal reached this targetrange.

TABLE 1 Drugs and Treatment Schedule Regimen 1 Regimen 2 Gr. N Agentmg/kg Route Schedule Agent mg/kg Route Schedule 1# 10 Vehicle — Po (5/2)× 3 — — — — 2 10 RX-3117 60 Po (5/2) × 3 — — — — 4 10 anti-PD-1 RMP1-14100* Ip biwk × 2 — — — — 5 10 RX-3117 60 Po (5/2) × 3 anti-PD-1 RMP1-14100* Ip biwk × 2 #—Control Group, *—μg/animal

TABLE 2 Tumor Growth Inhibition and Survival Benefits of CombiningRX-3117 with a PD-1 Inhibitor TGI Day Gr. Treatment Group 28 PR CR TFS 1Vehicle — 2 0 0 2 RX-3117 (60 mg/kg) 80% 0 0 0 3 anti-PD-1 RMP1-14 (100μg) 93% 1 3 2 4 RX-3117 + anti-PD-1 99% 2 7 7 TGI: Tumor growthinhibition; at Day 28; PR: No. of Partial Regressions; CR: No. ofComplete Regressions; TFS: No. of Tumor Free Survivors; all at Day 45

Tumors were measured in two dimensions using calipers, and volumecalculated using the formula:

${{Tumor}\mspace{14mu} {{Volume}\left( {mm}^{3} \right)}} = \frac{w^{2} \times l}{2}$

where w=width and 1=length, in mm, of the tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Treatment efficacy was determined using data from Day 45. The MTV (n),the median tumor volume for the number of animals, n, on Day 45, wasdetermined for each group. Percent tumor growth inhibition (% TGI) isdefined as the difference between the MTV of the designated controlgroup (vehicle administration) and the MTV of the drug-treated group,expressed as a percentage of the MTV of the control group:

${\% \mspace{14mu} {TGI}} = {{\left( \frac{{MTV}_{control} - {MTV}_{{drug}\text{-}{treated}}}{{MTV}_{control}} \right) \times 100} = {\left\lbrack {1 - \left( {{MTV}_{{drug}\text{-}{treated}}/{MTV}_{control}} \right)} \right\rbrack \times 100}}$

The data set for TGI analysis includes all animals in a group, exceptthose that died due to treatment-related (TR) or non-treatment-related(NTR) causes. An agent that produces at least 60% TGI in this assay isconsidered to be potentially therapeutically active.

The study protocol specifies a tumor growth delay assay based on themedian time to endpoint (TTE) in a treated group versus the controlgroup. Each animal was euthanized for tumor progression (TP) when itstumor reaches the 1500 mm³ volume endpoint. The time to endpoint (TTE)for each mouse is calculated with the following equation:

${TTE} = \frac{{\log_{10}\left( {{endpoint}\mspace{14mu} {volume}} \right)} - b}{m}$

where b is the intercept and m is the slope of the line obtained bylinear regression of a log-transformed tumor growth data set. The dataset is comprised of the first observation that exceeds the studyendpoint volume and the three consecutive observations that immediatelyprecede the attainment of the endpoint volume. Any animal that did notreach endpoint was euthanized at the end of the study and assigned a TTEvalue equal to the last day of the study (71 days). In instances inwhich the log-transformed calculated TTE precedes the day prior toreaching endpoint or exceeds the day of reaching tumor volume endpoint,a linear interpolation is performed to approximate TTE. Any animaldetermined to have died from treatment-related (TR) causes is assigned aTTE value equal to the day of death. Any animal that died fromnon-treatment-related (NTR) causes is excluded from TTE analysis.

Treatment efficacy was determined from the number of regressionresponses. Treatment may cause partial regression (PR) or completeregression (CR) of the tumor in an animal. In a PR response, the tumorvolume is 50% or less of its D1 volume for three consecutivemeasurements during the course of the study, and equal to or greaterthan 13.5 mm³ for one or more of these three measurements. In a CRresponse, the tumor volume is less than 13.5 mm³ for three consecutivemeasurements during the course of the study. Any animal with a CRresponse on the last day of the study was additionally classified as atumor-free-survivor.

For toxicity assessments, animals were weighed daily for the first fivedays of the study and twice weekly thereafter. The mice were observedfrequently for overt signs of any adverse, treatment-related sideeffects, and clinical signs of toxicity are recorded when observed.

Acceptable toxicity is defined as a group mean body-weight loss of lessthan 20% during the study and not more than one treatment-related (TR)death among ten treated animals. Any dosing regimen resulting in greatertoxicity is considered above the maximum tolerated dose (MTD). A deathis classified as TR if attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or if due to unknown causesduring the dosing period or within fourteen days of the last dose. Adeath is classified as non-treatment-related (NTR) if there is noevidence that death was related to treatment side effects.

Prism 6.05 (GraphPad) for Windows was employed for statistical andgraphical analyses. MTV values for multiple groups are compared with thenon-parametric Kruskal-Wallis test and a post hoc Dunn's multiplecomparison test. The two-tailed statistical analyses were conducted atP=0.05. Prism reports results as non-significant (ns) at P>0.05,significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”)at 0.001<P≤0.01 and extremely significant (“***”) at P≤0.001. Becausestatistical tests are tests of significance and do not provide anestimate of the size of the difference between groups, all levels ofsignificance are described as either significant or non-significantwithin the text of this report.

A “box and whiskers” diagram was constructed to show the distribution ofindividual tumor volumes, by group, on D15. The box represents the25^(th) to 75^(th) percentile of observations, the horizontal linecorresponds to the median value, and the “whiskers” indicate the maximumand minimum values. Group median tumor volumes were plotted as functionsof time. Group mean BW changes are graphed as percent change, ±SEM, fromD1. Animals that died from NTR causes are excluded from all graphicalpresentations.

Survival was analyzed by the Kaplan-Meier method, based on TTE values.The logrank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests determine thesignificance of the difference between the overall survival experiences(survival curves) of two groups, based on TTE values. The Kaplan-Meierplot and statistical tests share the same data sets, and exclude anyanimals that are recorded as NTR deaths. A scatter plot is constructedto show TTE values for individual mice, by group; this plot shows NTRdeaths, which are excluded from all other figures. Group mean tumorvolumes are plotted as functions of time. When an animal exits the studybecause of tumor size or TR death, its final recorded tumor volume isincluded with the data used to calculate the median volume at subsequenttime points. Tumor growth curves are truncated after two TR deaths occurin the same group. Group mean BW changes over the course of the studyare graphed as percent change, ±SEM, from Day 1. Tumor growth and BWchange curves are truncated after more than half the assessable mice ina group exits the study.

Example 3: Pharmacokinetics, Safety and Tolerability of RX-3117 inHumans

In a first-in-human, open-label, exploratory study, thepharmacokinetics, safety and tolerability of RX-3117 were evaluated. Thestudy duration was 14-15 days (7-day screening period; 3-day treatmentperiod; 4 (+1)-day safety follow-up period). Nine adult male and femalesubjects with histologically confirmed, solid tumors enrolled in andcompleted the study. The subjects received RX-3117 (n=3 subjects perdose) as a single oral dose (50 mg or 100 mg) or a single intravenousdose (20 mg).

Pharmacokinetics (PK)

The absolute bioavailability (F) for oral RX-3117 was 55.67% and 33.42%for the 50 and 100 mg doses, respectively. The mean T_(max) was 2.16hours and 2.49 hours for the 50 and 100 mg doses, respectively. The meanC_(max) was 303.3 ng/mL and 311.43 ng/mL for the 50 and 100 mg doses,respectively. The greater absolute bioavailability and C_(max) resultsof the 50 mg dose compared to the 100 mg dose suggests that oralbioavailability of RX 3117 in plasma may not be dose-proportional. TheT_(1/2) for the 50 mg and 100 mg doses was 13.95 hours and 20.92 hours,respectively, indicating that RX-3117 may show dose proportionality onsome parameters but not on others at the doses tested.

The plasma PK profile of intravenous RX-3117 differed from the plasma PKprofile of oral RX-3117. The 20 mg dose of intravenous RX-3117 recoveredrapidly after bolus infusion (T_(max)=0.25 hours). The 20 mg dose ofintravenous RX-3117 had a mean C_(max) of 1143.63 ng/mL, which wasapproximately a 4-fold increase over the peak concentrations of the oraldoses.

Safety and Tolerability

RX-3117 was safe and well-tolerated in all subjects. No adverse event(AE), treatment-emergent adverse event (TEAE) or serious adverse event(SAE) occurred.

The results show that RX-3117 is safe and well-tolerated with oralbioavailability, and support the study of higher doses.

Example 4: Pharmacokinetics, Safety and Tolerability of RX-3117 atDifferent Oral Doses

In an open-label, dose-ranging study, the pharmacokinetics (PK) ofRX-3117 at various oral doses was evaluated. Subjects with advancedmalignant tumors were administered capsules containing RX-3117 at dailydoses of 30 mg (N=1), 60 mg (N=1), 100 mg (N=3), 150 mg (N=3), 200 mg(N=3), 500 mg (N=3), 1000 mg (N=3), 1500 mg (N=4), and 2000 mg (N=5 todate) 3 times a week (TIWK) for 3 weeks with 1 week off during each 4week cycle. Based on the continued safety profile, and to enhance weeklyRX-3117 exposure, more frequent dosing was also implemented. In additionto the TIWK dosing scheme discussed above, subjects also received 500 mgand 700 mg 5 times a week, and 500 mg for 7 times a week for 3 weekswith 1 week off during each 4 week cycle. Dose escalation began with anaccelerated design treating 1 subject per dose (Simon et al., J. Natl.Cancer Inst., 89(15): 1138-47 (1997) followed by a standard 3+3 designusing a modified Fibonacci sequence after the occurrence of a singlerelated Grade 2 or greater adverse event. Table 3 summarizes the dosingschedule.

TABLE 3 Dose Escalation - 3 Times per Week Dose Actual dose Total weeklyTotal cycle Group (mg) Frequency dose (mg) dose (mg) 1 30 3 times perweek 90 270 2 60 3 times per week 180 540 3 100 3 times per week 300 9004 150 3 times per week 450 1,350 5 200 3 times per week 600 1,800 6 5003 times per week 1,500 4,500 7 1,000 3 times per week 3,000 9,000 81,500 3 times per week 4,500 13,500 9 2,000 3 times per week 6,00018,000 10 500 5 times per week 2,500 7,500 11 700 5 times per week 3,50010,500 12 500 7 times per week 3,500 10,500

Pharmacokinetics (PK)

PK data are presented in Table 4.

RX-3117 was rapidly absorbed without a marked lag time, with medianT_(max) usually observed at 2 to 3 hours. After T_(max), elimination wasbiphasic with about half of AUC_(0-t) (0-24 hours) observed in the first8 hours, and over 80% by 24 hours. Apparent terminal T_(1/2) did notexhibit dose-dependent or time-dependent pharmacokinetics, with meanvalues over the dose range 60 to 2000 mg ranging from 11.6 to 16.7 hoursafter the first dose, and from 12.3 to 20.2 hours after the seventh dose(Day 15 of dosing). C_(max) and AUC_(0-t) (0-24 hours) increased fairlylinearly with dose, but in a less than proportional manner, possiblyreaching a plateau by the 1500 mg dose (FIGS. 12 and 13). Over the doserange of 30 to 2000 mg, mean C_(max) ranged from 32 to 1858 ng/mL afterthe first dose, and from 99 to 1703 ng/mL after the seventh dose (FIG.12). Over the same dose range, mean AUC_(0-t) (0-24 hours) ranged from164 to 20,544 hr·ng/mL after the first dose, and from 702 to 20,919hr·ng/mL after the seventh dose (FIG. 13). Accumulation was generallyminimal.

The PK data show a dose dependent increase in exposure with doses up to1000 mg TIWK. At doses greater than 500 mg TIWK, the C_(max) andAUC_(0-t) (0-24 hours) after the 7th dose are consistently lower thanthose measured after the first dose (FIGS. 12 and 13). Due to theplateauing of C_(max) and AUC_(0-t) (0-24 hours) values at doses above1000 mg, a more frequent dosing schedule was used to enhance weeklyexposures (Table 4). Based on the results of this study the maximumtolerated dose (MTD) for RX-3117 was determined to be 700 mg daily at 5days per week, given for three weeks with 1 week off per 4-week cycle.This MTD was then used for follow-up efficacy studies.

TABLE 4 PK Data after Days 1 and 15 of Study Dose Dose Dose C_(max)T_(max) T_(1/2) AUC₀₋₂₄ Frequency (mg) Day Number (ng/mL) (hr) (hr)(hr*ng/mL) 3 per Week 30 1 1 31.6 2 3.87 252 3 per Week 60 1 1 139 216.2 1164 3 per Week 100 1 1 357 2 15.4 2714 3 per Week 150 1 1 511 313.9 3546 3 per Week 200 1 1 637 2 13.3 4719 3 per Week 500 1 1 1104 216.7 7916 3 per Week 1000 1 1 1635 2 11.6 12218 3 per Week 1500 1 1 16223 11.8 15322 3 per Week 2000 1 1 1858 3 13.3 17044 5 per Week 500 1 11441 2 7.31 12373 5 per Week 700 1 1 989 3 9.05 8663 7 per Week 500 1 11269 3 8.28 10097 3 per Week 30 15 7 98.9 2 8.23 702 3 per Week 60 15 7113 4 15.7 1566 3 per Week 100 15 7 460 2 20.2 3289 3 per Week 150 15 7360 3 15.1 2437 3 per Week 200 15 7 643 3 16.2 4574 3 per Week 500 15 7941 3 15.3 8275 3 per Week 1000 15 7 1210 3 14.9 9753 3 per Week 1500 157 883 2 13.8 7050 3 per Week 2000 15 7 1703 3 12.1 17403 5 per Week 50015 11 1212 2 8.71 9201 5 per Week 700 15 11 674 4 11.2 6321 7 per Week500 15 15 1363 2.5 9.45 14467

Safety and Tolerability

The most frequently observed adverse events were mild to moderatefatigue and nausea, mild diarrhea, mild vomiting, mild anorexia andmoderate dehydration. Dose limiting toxicities were limited to Grade 3anemia, thrombocytopenia.

Example 5: Efficacy, Safety and Tolerability of RX-3117 in Humans

The efficacy, safety and tolerability of RX-3117 at various doses andfrequencies were evaluated (see Example 4, above). Subjects withadvanced malignant tumors were administered capsules containing RX-3117at various doses, from TIWK to 7 times per week for 3 weeks with 1 weekoff during each 4-week cycle. Dose escalation begins with an accelerateddesign treating 1 subject per dose (Simon et al., J. Natl. Cancer Inst.,89(15):1138-47 (1997) followed by a standard 3+3 design using a modifiedFibonacci sequence after the occurrence of a single related Grade 2 orgreater adverse event. Table 4 (above) summarizes the dosing schedule.

The subjects were assessed for efficacy, safety and tolerability ofRX-3117. Total of 48 subjects were enrolled (30 Females, 18 males).Seventeen subjects experienced stable disease for 1 to 10 cycles; with10 subjects receiving treatment from 82 to 276 days. A tumor burdenreduction was seen in 3 subjects with pancreatic (tumor volume andbiomarkers of CA19-9), breast and mesothelioma cancers. The mostfrequent related adverse events were moderate to severe anemia, mild tomoderate fatigue and nausea, mild diarrhea, vomiting, and anorexia.

In another stage of this study, RX-3117 is being evaluated in a PhaseIb/IIa clinical trial in cancer patients with relapsed or refractorypancreatic cancer or advanced bladder cancer (including muscle-invasivebladder cancer). The Phase Ib/IIa clinical trial is a multi-center studythat evaluates the safety and efficacy of RX-3117 in these targetpatient populations. Secondary endpoints include safety andpharmacokinetic analyses. Patients in the trial are receiving a dailyoral dose of RX-3117 of 700 mg, five times weekly for three weeks in a28 day cycle and 4 treatment cycles, or until their disease progresses.

Example 6: The Radiosensitizing Effect of Fluorocyclopentenyl-Cytosine(RX-3117) in Ovarian and Lung Cancer Cell Lines Drugs and Chemicals

Stock solutions of RX-3117 were made in deionized water. All otherchemicals used were of standard quality and commercially available.

Cell Culture

The human NSCLC cell lines A549 (adenocarcinoma), H460 (large cellcarcinoma), SW1573 (alveolar carcinoma), and SW1573/G- (SW1573 cell lineresistant to gemcitabine) and the ovarian cancer cell line A2780 werekept in exponential growth in T25 flasks (Greiner Bio-One GmbH,Frickenhausen, Germany) and cultured in Dulbecco's minimal essentialmedium (DMEM) or RPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum (FBS), 1% streptomycin and penicillin, and maintained at37° C. under a saturated atmosphere of 5% CO₂. Cells were harvestedusing trypsin EDTA (Invitrogen, Paisley, UK). A Coulter® Z™ seriescounter was used to count cells.

Clonogenic Assay

Exponential growing A2780, SW1573, A549, H460 and SW1573/G- cells wereexposed to 1 μM RX-3117 or untreated (control) for 24 h and irradiatedwith single doses γ-radiation (0-6 Gy) using a ⁶⁰Co source (Gammacell200, Atomic Energy of Canada, Ltd). Subsequently, 500 cells/T25 flaskswere plated and allowed to form colonies. After ten days colonies werefixed with 100% ethanol and stained with 10% Giemsa stain solution(Merck Chemicals BV, Amsterdam, the Netherlands) for colonies counting.Plating efficiency (PE) was calculated by dividing the number ofcolonies formed through the number of cells plated and normalized forcytotoxicity induced by control. To illustrate the effect of RX-3117 onradiation, dose modifying factor (DMF) was calculated as describedearlier (Bijnsdorp I V, van den Berg J, Kuipers G K, Wedekind L E,Slotman B J, van Rijn J, Lafleur M V M and Sminia P: Radiosensitizingpotential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicamon human glioma cells. J Neurooncol 85: 25-31, 2007).

Spheroid Assay

The NSCLC cell lines A549 and SW1573 were plated in low attachment 24well plates (Corning Incorporated, Corning, N.Y.) at a density of100,000 cells/well and allowed to form spheroids. After three days,single spheroids were transferred to new 24 well low attachment plates(one spheroid/well). Immediately after transfer treatment was started,for A549 and SW1573 cells 1 μM RX-3117 was combined with fractionated 2Gy irradiation (5 days single 2 Gy dose). Pictures were taken on day 0(before irradiation) and after 3, 6, 9, and 15 days using a phasecontrast microscope (LeicaDMI300B Universal Grab 6.3 software, DigitalCell Imaging Labs). The measurements were taken by ImageJ software(ImageJ 1.45s, Wayne Rasband, National Institutes of Health, Bethesda,Md.) for spheroid volume calculation (V=4/3π(D/2)³) as described earlierby Galvani et al. (Galvani E, Giovannetti E, Saccani F, Cavazzoni A,Leon L G, Dekker H, Alfieri R, Carmi C, Mor M, Ardizzoni A, Petronini PG and Peters G J: Molecular mechanisms underlying the antitumor activityof 3-aminopropanamide irreversible inhibitors of the epidermal growthfactor receptor in non-small cell lung cancer. Neoplasia 15: 61-72,2013).

Flow Cytometry Analysis

Cell cycle distribution and apoptosis were analyzed by plating cells inflat bottom 6 well plates (Greiner Bio-One GmbH, Frickenhausen, Germany)at the density of 5,000 cells and allowed to attach for 24 h. Thereaftercytotoxic concentrations of 5×IC₅₀ were added. The exposure time was 24h and 48 h and for comparison the control group was included. At eachtime point total amount of adherent and floating cells were harvested inround-bottom FALCON tubes (BD, Franklin Lakes, N.J., USA). Aftercentrifugation, cell pellets were resuspended in 1.0 ml propidium iodide(PI) solution (50 ug/ml PI, 0.1% sodium citrate 0.1% Triton X-100, 0.1mg/ml ribonuclease A) or 10 ul Annexin V (cat#31490014, Immunotools) andleft on ice for 30 minutes. Subsequently, samples were analyzed usingFACSCalibur (BD Biosciences, Mount View, Calif., USA). For data analysisCELLQuest™ software was carried out, using gates on DNA histograms toestimate the amount of cells in sub-G1 phase (apoptotic cells).

Protein Expression Analysis

The influence of RX-3117 on protein expression during differenttreatment conditions was analyzed by western blot. Cells were lysedusing 1× cell lysis buffer (Cell Signaling, Danvers, Mass., USA)containing 4% protease inhibitor cocktail (Roche Diagnostics, Mannheim,Germany) on ice for 30 minutes and centrifuged for 10 minutes at 4° C.at 14,000 rpm. Bio-Rad assay was performed to determine protein amountin the collected supernatant as described earlier (Lemos C, Kathmann I,Giovannetti E, Calhau C, Jansen G and Peters G J: Impact of cellularfolate status and epidermal growth factor receptor expression onBCRP/ABCG2-mediated resistance to gefitinib and erlotinib. Br J Cancer100: 1120-7, 2009). The following antibodies were used for proteinexpression: γH2A.X (cat#9718, Cell Signaling, 1:1000), β-actin (Sigma,1: 10,000), Cdc25C Ser216 (cat#4901, Cell Signaling, 1:1000), Cdk1 Tyr15(cat#9111, Cell Signaling, 1:1000), Chk1 Thr68 (cat#2197S, CellSignaling, 1:1000), Histone 3 (cat#4499, Cell Signaling). Antibodieswere diluted in 1:1 solution with Rockland buffer (Rockland Inc,Philadelphia, Pa.) and phosphate buffered saline (PBS) supplemented with0.05% Tween 20. Proteins were separated in 20% SDS-PAGE gel andtransferred to polyvinylidene difluoride (PVDF) membrane. Forfluorescent signal secondary anti-bodies goat anti-mouse InfraRedDye andgoat anti-rabbit InfraRedDye were used. Proteins were detected by anOdyssey InfraRed Imager (Li-COR Bioscience, Lincoln, Nebr.).

Results

Radiosensitizing Effect of RX-3117

To investigate the effect of RX-3117 on radiation, clonogenic assayswere performed. First, the inventors examined whether pre- orpost-incubation with RX-3117 enhanced the effect of radiation. Pre- andpost-treatment with RX-3117 together with 4 Gy were compared in A2780cells.

The clonogenic assay data showed that pre-incubation with RX-3117 wasthe most effective condition for a radiosensitizing effect.Pre-incubation with 1 μM RX-3117 had a five times lower platingefficiency compared to control (FIG. 15).

Using the pre-incubation schedule, the potential radiosensitizing effectwas investigated in A2780 cells and the non-small cell lung cancer celllines A549, SW1573 and SW1573/G- and H460. In general, all cell linesshowed a radiosensitizing effect when treated with RX-3117 andradiation. The greatest radiosensitizing was observed in the A2780 andA549 cell lines with a DMF of 1.8 and SW1573 with DMF of 1.5 (FIGS. 16A,16B, 16D). The gemcitabine resistant cell line SW1573/G- had a DMF of1.4 (FIG. 16E), but H460 cells showed a poor radiosensitizing effect.Since fractionated radiation is the standard procedure in the clinic, afractionated dose of 2 Gy irradiation during 5 days in SW1573 cells wasalso studied. Incubation with 1 μM of RX-3117 prior to fractionatedradio therapy of 5 times 2 Gy showed the lowest colonies outgrowth (FIG.16F).

The radiosensitizing ability of RX-3117 in combination with irradiationin a 3-dimensional model using a spheroid assay was also investigated.The sphere formation assay revealed a radiosensitizing effect of RX-3117on spheres in SW1573 and A549 spheroids (FIG. 17). SW1573 spheres werehighly affected by both 1 μM RX-3117 alone and by radiotherapy (RT)alone (2 Gy 5 days) and the effect was enhanced with the combination. InA549 cells, RX-3117 treatment or irradiation alone had only a smalleffect on the volume growth while 1 μM RX-3117 enhanced the effect of 5days irradiation with 2 Gy (FIG. 17).

Apoptosis Initiation

The potential of RX-3117 to induce apoptosis was studied in the NSCLCcell lines. The amount of apoptotic cells were measured by Annexin Vstaining (FIG. 18) after 24 h, 48 h and 72 h of exposure. Annexin V cellmembrane staining showed a gradual increase in apoptotic cells for A549cells and SW1573 cells, which was more pronounced for SW1573 cells thanfor A549.

DNA Damage Initiation

DNA damage is a hallmark for cell death. DNA damage was studied byevaluation of γH2A.X expression. In the A2780 cell line graduallyincreasing RX-3117 concentrations starting by 0.1 μM to 10 μM RX-3117showed induction of γH2A.X S139 in a dose dependent manner (FIG. 18A).In the SW1573 cell line the double strand break damage marker wasincreased after 48 h of exposure to 0.3 μM of RX-3117 (FIG. 18B). Acombination of 0.3 μM of RX-3117 and irradiation showed more pronouncedγH2A.X S139 protein expression (FIG. 18B). As expected radiation causedan immediate increase of the expression of γH2A.X; in the presence ofRX-3117 the repair was delayed.

Effect of Treatment with RX-3117 and Radiation on Cell CycleDistribution and Cell Death

Since a disturbance in cell cycle distribution has been reported to beimplicated in the radiosensitizing effect of other nucleoside analogs(Shewach D S and Lawrence T S: Antimetabolite radiosensitizers. J ClinOncol 25: 4043-50, 2007), the effect of RX-3117 in combination withradiation was investigated using FACS analysis in the NSCLC cells. Atthe relatively low concentration of 1 μM RX-3117 a small but justsignificant (p<0.05) cell line dependent increase of the S-phase wasfound in 3 out of 4 cell lines. In all cell lines an increase in the G1phase was found (FIG. 19A) and a strong decrease of the G2/M phase wasfound. Radiation at 4 Gy caused a clear decrease of the amount of cellsin the S-phase, an increase in the G2/M phase in both SW1573 cells andno effect in A549 cells, but a decrease in the H460 cells (FIG. 19A).The combination of radiation and RX-3117 led to an increased number ofcells in the S-phase in both SW1573 variants, but a decrease in H460cells. In A549 cells cell kill (sub G1) was clearly increased, but notin the other cells (FIG. 19A).

In order to understand some of these phenomena the effect of RX-3117 andradiation on the expression of some essential cell cycle proteins wasalso investigated. (FIG. 19B, and FIG. 20). In SW1573 the effect of bothRX-3117 and radiation were examined on various cell cycle checkpointproteins (FIG. 20). Radiation caused an interesting decrease in wee1,Chk2, CDC25c and p-CDC25c after 48 hr. In both SW1573 cells radiationcaused an increase in the phosphorylation of Chk2 (FIG. 19B). In almostall cell lines (except the gemcitabine resistant SW-1573/G) RX-3117decreased the phosphorylation of Cdk1; similarly radiation increased thephosphorylation of cdc-25C (but not in SW1573/G-) in 24 hr (FIG. 19B).In combination with RX-3117 this effect was maintained (FIG. 19B).

Example 7: Inhibition of DNA Methyltransferase by RX-3117 Leads toUpregulation of Hypomethylated Targets

RX-3117 resembles azacytidine (aza-CR) and aza-deoxycytidine (aza-CdR).RX-3117 is taken up by the human equilibrative nucleoside transporter(hENT) and activated by uridine-cytidine kinase 2 (UCK2) to RX-3117-MP(FIG. 21). RX-3117 is taken up by the human equilibrative nucleosidetransporter (hENT) and activated by uridine-cytidine kinase 2 (UCK2) toRX-3117-MP. RX-3117 downregulates DNA methyltransferase 1 (DNMT1) (ChoiW. J., et al., J. Med. Chem. 55 (2012) 4521-4525; Peters G. J., et al.,Invest New Drugs 31 (2013) 1444-1457). DNMT1 is responsible formaintaining methylation in newly synthesized DNA in the S-phase andmethylates cytosine residues in hemimethylated DNA. The rate ofdeamination of RX-3117 is much slower than gemcitabine.

RX-3117 is an orally bioavailable novel cytidine analog which iscurrently being evaluated in Phase I clinical study. The maximaltolerated dose is higher than 2,000 mg/day. Downregulation of DNMT1 byRX-3117 has been shown in various cell lines with different histologicalbackgrounds. Currently both UCK2 and DNMT1 are being evaluated aspotential biomarkers. In this example, the effect of RX-3117 on DNMT1 atthe DNA, RNA, protein and enzyme activity, and reactivation ofsuppressed target genes, including p16INK4A, methylguaninemethyltransferase (MGMT) and the proton coupled folate transporter(PCFT) were determined. PCFT includes transports folic acid,methotrexate (MTX) and pemetrexed (PMX) at pH 5.5 and 7.4, and the geneis highly methylated. In addition, the function of proteins for whichthe gene is known to be regulated by methylation are studied, including:proton-coupled folate transporter (PCFT). Expressions of E-cadherin (anadhesion molecule), p16INK (a tumor suppressor protein), and 0-6Methylguanine DNA methyltransferase (MGMT) (a DNA repair gene) in A549cell line were also studied.

Methods

In this study, the following cell lines were used: (1) CCRF-CEM cellsand its MTX resistant variant CEM-MTX, characterized by a deficiency ofthe reduced folate carrier (RFC) (Jansen G., et al., JBC 273 (1998)30189-30198). The PCFT gene in CEM cells is highly methylated. (GonenN., et al., BBRC 376 (2008) 787-92); (2) CEM cells cultured in RPMImedium with 10% fetal bovine serum (FBS); and (3) A549 and SW1573non-small cell lung cancer (NSCLC) and A2780 ovarian cancer cell linescultured in DMEM medium with 10% FBS.

DNMT1 protein expression was measured by Western Blotting after exposureto RX-3117 for 24 or 48 hr. DNMT1 RNA expression was measured byreal-time PCR after 24 and 48 hr exposure to RX-3117. DNMT enzymeactivity was measured in isolated nuclei after exposure 1 μM RX-3117 or5 μM aza-CdR using a DNA methyltransferase assay kit provided byEpiGentek using the ability of a CpG dinding domain to bind tomethylated DNA. In A549 cells the effect of 5 μM RX-3117 on overallmethylation was measured with a specific antibody against5-methyl-cytosine. Bands on Western blots were visualized usingappropriate InfraRedDye using an Odyssey InfraRed imager.

MTX transport was measured using radiolabeled MTX in CEM wild type andCEM-MTX cell lines. CEM cells have a high RFC activity. CEM-MTX arecompletely deficient in RFC-mediated transport. CEM cells have a highlymethylated PCFT transporter and a very low PCFT mediated transport(Gonen N., et al., BBRC 376 (2008) 787-92). MTX transport at pH 7.4 ispredominantly RFC mediated and less than 2% by PCFT. Folic acid was usedto inhibit PCFT mediated transport. L-leucovorin (L-LV) was added tocompletely inhibit RFC mediated transport. CEM and CEM-MTX cells wereexposed to 29.6 μM RX-3117 and to 0, 19 μM aza-CdR as a positivecontrol. MTX transport was measured after 24 hr to the drugs in a 3minutes uptake assay using 2 μM [3′,5,′7-3H]-MTX.

Statistics were done using the Student's t-test.

Results

In the moderately sensitive non-small cell lung cancer (NSCLC) celllines such as A549 and SW1573, 5-50 μM RX-3117 downregulated DNMT1protein expression by 5-20% after 24 hour exposure and >90% after 48 hr(FIGS. 25A and B). DNMT1 mRNA was not affected after 24 hours exposurebut was affected moderately after 48 hr (FIGS. 25 C and D).

In the sensitive ovarian cancer cell line A2780, protein down regulationwas already observed after 24 hr at 1 μM RX-3117 (FIGS. 26 A and B).DNMT1 activity was inhibited by 1 μM RX-3117 by 32% which was similar tothe percent inhibition with 5 μM of the reference compound5-aza-2′-deoxycytidine (DAC, 31%).

In A549 cells, 5 μM RX-3117 decreased overall methylation of DNA(detected by an antibody against 5-methylcytosine) by 25% after 48 hrexposure, while 5 μM DAC only inhibited 9% (FIG. 27A). For several genesknown to be affected by methylation, protein expression and activitywere evaluated. A549 cells were exposed to RX-3117 and measured usingimmunofluorescence with an antibody against 5-methyl-cytosine (FIG.27B). In A549 and SW 1573 cells a 24 hr exposure to 5 μM RX-3117increased the expression of the cell cycle protein p16INK4A and of theDNA repair enzyme MGMT. FIG. 27C shows the expression of MGMT,E-cadherin, and p16INK4 after exposure to RX-3117 and aza-dC.

For PCFT, functional activity of RX-3117 was evaluated in CCRF-CEMleukemic cells which have a highly methylated PCFT promoter and inCEM-MTX cells which are deficient for the reduced folate carrier (RFC).PCFT is a specific folate transporter responsible for uptake of folicacid and the folate analogs methotrexate (MTX) and pemetrexed.Incubation of both CEM and CEM-MTX cells with either 29.6 μM RX-3117 orDAC as a positive control markedly increased PCFT mediated transport ofMTX. This was more pronounced in CEM-MTX cells, 10-11-fold increase forboth RX-3117 and DAC, compared to a 4-fold increase in CEM cells. Folicacid (FA) was added to inhibit PCFT and L-LV to inhibit RFC mediated MTXtransport. Aza-CdR and Aza-CR were included as a positive control.(FIGS. 28 A, B and C).

Conclusion

In conclusion, RX-3117 downregulates DNMT1 protein and RNA expression bydecreasing DNA methylation. RX-3117 mediated hypomethylation increasesthe expression of MGMT, E-cadherin, PCFT, and the tumor suppressor genep16INK4A. PCFT mediated transport of MTX. These data underline DNMT1inhibition as a novel mechanism of RX-3117. RX-3117 is a new epigeneticmodulator.

Example 8: Evaluation of UCK2 Protein Expression as a PotentialPredictive Biomarker of RX-3117 Background

A novel, orally bioavailable nucleoside analogue, RX-3117, is a prodrugactivated intracellularly by Uridine Cytidine Kinase 2 (UCK2) that isthought to be expressed predominantly in tumor tissue. RX-3117 iscurrently being evaluated in a Phase Ib/IIa multi-center, open-labelclinical study in patients with advanced pancreatic and bladder cancer.In this study, the relation between UCK2 tissue protein expression andthe efficacy of RX-3117 in mice xenograft models and also UCK2 proteinexpression in a panel of human cancer tissues relative to normal tissuewere studied.

Methods

The UCK2 protein expression in tumor tissues was analyzed byimmunoblotting using clone 22-1 rabbit monoclonal antibody. Thevalidated procedure for the immunohistochemistry (IHC) of UCK2 withclone 22-1 was performed in a panel of human formalin-fixedparaffin-embedded (FFPE) cancer and normal tissues.

Results

The immunoblotting protein level of UCK2 normalized to beta-actin andcorresponding tumor growth inhibition (oral RX-3117 dose of 500 mg/kg,TIWK) were 57 and 67% in MiaPaCa2, 30 and −5% in BxPC3, 199 and 92% inColo-205, 21 and 90% in Caki-1, 2 and 39% in A549, and 146 and 79% inH460, respectively. These data indicate an anti-tumor efficacy trend ina UCK2-dependent manner. The IHC of UCK2 showed that positive stainingof UCK2 in cancer tissues was observed in 20/20 bladder cancer tissues(100% frequency), 19/20 CRC tissues (95% frequency), 18/20 NSCLC tissues(90% frequency), and 19/20 pancreatic cancer tissues (95% frequency).Average H-Scores of UCK2 in cancer tissues vs. normal tissues were 104vs. 9 in lung, 97 vs. 20 in bladder, 67 vs. 41 in pancreas and 39 vs. 21in colon, respectively.

Conclusions

The current data showed a correlation trend between UCK2 proteinexpression level and degree of antitumor activity of RX-3117 inxenograft models. It also supports a higher UCK2 protein expressionlevel in human cancer tissues compared to their normal tissues. Thissuggests that RX-3117 activity may be specific to tumor tissue, andquantification of UCK2 expression in human cancer tissues may be usefulas a predictive biomarker to select patients for their sensitivity toRX-3117 in future clinical studies.

Example 9: Synthesis of RX-3117 Monohydrate

Preparation of INT14 from ASM11 by continuous reaction of stage 1 to 3in fixed reactors ASM 11,tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane,(37.65 kg, 1 wt, 1 eq, 55 mol) was dissolved in 2-methyl tetrahydrofuran(4.0 vol, 3.4 wt). TBAF (tetra-n-butylammonium fluoride) 1.0 μM in THF(tetrahydrofuran, (1.61 vol, 1.45 wt, 1.1 eq.) was added to the reactionvessel in one portion (mild exotherm addition controlled) over 15 to 45min, maintaining 18 to 23° C. 2-Methyl tetrahydrofuran (1.0 vol, 0.9 wt)was charged to the vessel as a line rinse maintaining 18 to 23° C. andthe resulting solution stirred at 18 to 23° C. for 6 hr until completeby ¹H NMR. The reaction mixture was charged with 8% w/w sodium hydrogencarbonate (3.0 vol) and stirred at 18 to 23° C. for 5 to 10 min(caution: mild exotherm) and allowed the phases to separate and removethe lower aqueous phase (2×2.0 vol). The first 8% w/w sodium hydrogencarbonated extraction gave a milky aqueous layer and extended settletime did not clear the emulsion. Investigations showed the emulsion wasconfined to the aqueous layer and had a low organic content, thus theprocess was continued. The total separation of the first extraction was5 hours 29 minutes. The second 8% w/w sodium hydrogen carbonateextraction separated without issue taking only 52 minutes. The aqueousphase was extracted with 2-methyl tetrahydrofuran (2.0 vol, 1.7 wt) andline was rinsed with 2-methyl tetrahydrofuran (2.0 vol, 1.7 wt). Thecombined organic phase that contained INT12 was heated to 40 to 50° C.and concentrated to ca. 4 vol at 40 to 50° C. under reduced pressure.Sampling was performed for analysis and analyzed by Karl-Fischer untilwater content was ≤0.2% w/w. The process yielded a 158.8 kg net weightof INT12 ASM11-alcohol in 2-methyltetrahydrofuran containing 15.3% w/wINT12 ASM11-alcohol, equating to a 99.0% total yield. A 92.83% areapurity INT12 ASM11-alcohol ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol)was determined by HPLC analysis.

The INT12 solution was returned to the vessel, cooled to 0 to 5° C. andcharged with triethylamine (0.41 vol, 0.30 wt, 2.0 eq). After rinsingthe line with 2-methyltetrahydrofuran (1.0 vol, 0.9 wt), the solutionwas charged with methanesulphonyl chloride (0.17 vol, 0.25 wt, 1.5 eq)diluted in 2-methyl tetrahydrofuran (1.0 vol. 0.9 wt) (cautiously mix inthe header vessel) maintaining 0 to 5° C. over at least 30 min(Exothermic). Additional 2-methyl tetrahydrofuran (0.5 vol, 0.4 wt) wasadded as a line rinse maintaining 0 to 5° C. The contents of the vesselwere stirred at 0 to 5° C. until the reaction was complete by ¹H NMRafter 1 hour. The representative sample was removed after 1 hour andwould have been checked approximately every 2 hours thereafter ifnecessary from the reaction vessel and analyzed to check the remainingINT12. After checking 100% conversion by ¹H NMR analysis, water (4.0vol) was charged maintaining 0 to 10° C. and the reaction mixture warmedto 18 to 23° C. and stirred for 5 to 10 min at 18 to 23° C. The upperorganic phase in the vessel was separated and charged with 8% w/w sodiumhydrogen carbonate solution (4.0 vol) maintaining 18 to 23° C. Theresulting biphasic solution was stirred at 18 to 23° C. for 1 to 2 h andthe separated organic phase charged with 20% w/w aqueous ammoniumchloride (2.0 vol) and 2-methyl tetrahydrofuran (2.0 vol, 1.7 wt). Asrequired, the temperature wa adjusted to 18 to 23° C. The 20% w/wammonium chloride wash resulted in a vigorous gas evolution, probablydue to a reaction with residual sodium hydrogen carbonate from theprevious step. After stirring at 18 to 23° C. for 5 to 10 min and theupper organic phase was separated and charged with purified water (2.0vol) adjusted to 18 to 23° C. The separated organic phase that containedINT13 was concentrated under reduced pressure at 35 to 45° C. to ca. 2vol. Sampling was performed for analysis. The process yielded a 78.4 kgnet weight of INT13 ASM11-mesylate in 2-methyltetrahydrofuran containing34.9% w/w INT13 ASM11-mesylate, equating to a 94.9% total yield. A62.91% area purity INT13 ASM11-mesylate((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) was determined by HPLC analysis, with 32.23% areaTBDPS by-product present.

Continuously, the INT13 solution was charged with DMSO (3.8 vol, 4.2 wt)and heated to 40 to 45° C. to concentrate the organic phase at ≤45° C.under reduced pressure until no more solvent (2-methyltetrahydrofuran)distilled. The concentration was continued for 5 hours and 25 minutesand the IPC by ¹H NMR showed a 2-methyltetrahdrofuran content of 8.3%w/w. After cooling the solution to 27 to 33° C., cesium carbonate (1.2wt) and cytosine (0.41 wt) were charged. The reaction mixture was heatedto 33 to 37° C. and stirred until complete by HPLC. Sampling forN/O-alkylation ratio analysis was performed after 24 hours and atappropriate time points thereafter from the reaction vessel. After 33hours and 47 minutes the reaction was deemed complete with an IPC resultof 99.5% conversion. The ratio of the N- to O-isomers was at 99.03:0.97.On completion, the mixture was charged with isopropyl acetate (2.0 vol,1.7 wt) and purified water (4.0 vol, 4.0 wt) maintaining ≤50° C. (wateraddition is exothermic). After stirring for 5 to 15 minutes the biphasicmixture was allowed to settle for 10 minutes and then separated theupper organic phase. The aqueous phase was re-extracted two times torecover all the product with isopropyl acetate (2.0 vol, 1.7 wt each) bystirring at 40 to 50° C. for 5 to 15 min and again allowing settling for10 minutes before separating. The combined organic phase was cooled to25 to 30° C. and charged with 10% v/v acetic acid (3.0 vol) and 26% w/wbrine solution (1.0 vol) maintaining 25 to 30° C. and the biphasicsolution was stirred at 25 to 30° C. for 30 to 60 min. The upper organicphase was washed three times with 10% v/v acetic acid (3.0 vol) and 26%w/w brine solution (1.0 vol) maintaining 25 to 30° C. In each wash stepthe upper organic solution was sampled by ¹H NMR analysis. The organicphase was washed again with ca. 3% w/w brine solution (3×2.0 vol) at 25to 30° C. and sampled for acetic acid content by ¹H NMR. The organicphase that contained INT14 was heated to 35 to 45° C. and concentratedto ca. 5 vol at 35 to 45° C. under reduced pressure. The solution wascharged with isopropyl acetate (3.0 vol, 2.6 wt) and concentrated to ca.5 vol at 35 to 45° C. under reduced pressure. The solution was chargedagain with isopropyl acetate (5.0 vol, 4.4 wt), adjusted to 57 to 63°C., stirred at 57 to 63° C. for 1.5 to 3 h and checked by HPLC forcrystallization/precipitation. The slurry was cooled to 35 to 45° C. andconcentrated to ca. 5 vol at 35 to 45° C. under reduced pressure. Theslurry was cooled further to 18 to 23° C. over 1.0 to 2.0 h and chargedwith n-heptane (7.0 vol, 4.8 wt) maintaining 18 to 23° C. over 30 to 90min. After 1 to 2 h at 18 to 23° C. and 1 to 2 h at 0 to 5° C., theslurry was filtered through 20 m cloth and washed with premixedn-heptane/isopropyl acetate (5:1, 2×1.0 vol) at 0 to 5° C. The productthat contained INT14 was dried under vacuum at up to 55° C. and assayedby ¹H NMR. Pass criteria was ≤2.0% w/w isopropyl acetate and ≤2.0% w/wn-heptane. The process yielded a 24.27 kg net weight of INT14,4-amino-1-((3 aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one,(45 mol, 93.25% purity), equivalent to 82% total and 64% w/w yield.

Preparation of RX-3117 Monohydrate from INT14 by Continuous Reaction ofStage 4 to 5 in Fixed Reactors

INT14, 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one,(24.27 kg, 45 mol, 1.0 wt) was charged to a vessel followed by methanol(7.5 vol, 5.9 wt) and the temperature of the reaction mixture wasadjusted to 18 to 23° C. To the reaction vessel, 2 μM HCl (1.1 vol, 1.2eq) was added maintaining temperature <50° C. The slurry mixture washeated to 45 to 55° C. and stirred at 45 to 55° C. (target 50° C.) for 2to 2.5 h. The vessel volume was noted and the reaction mixture wasdistilled under reduced pressure maintaining 45 to 55° C. andmaintaining constant volume by the addition of MeOH (5.0 vol, 4.0 wt).The mixture was sampled and continued to charge MeOH (2.5 vol 2.0 wt)maintaining constant volume by distillation at 45 to 50° C. until ≤1.0%area acetonide intermediate was present by HPLC. Once the conversion wascompleted, the reaction mixture was allowed to cool to 25 to 30° C. Thereaction mixture was concentrated to 5 volumes under reduced pressuremaintaining 35 to 45° C. The reaction mixture was cooled to 25 to 30° C.To the reaction vessel was charged with TBME (methyl tert-butyl ether)(5.0 vol, 3.7 wt) and water (5.0 vol) maintaining 25 to 30° C. Thebi-phasic solution was stirred at 25 to 30° C. for 10 to 20 min and thephases were separated at 25 to 30° C. retaining the lower aqueous phase.The retained lower phase was transferred to the vessel and rechargedwith TBME (5.0 vol, 3.7 wt) maintaining 25 to 30° C. After stirring thebiphasic solution at 25 to 30° C. for 10 to 20 min, the lower aqueousphase was separated. The lower aqueous phase was returned to the vesseland line was rinsed with water for injection (0.5 vol, 0.5 wt). Theremoval of trityl alcohol was checked by ¹H NMR assay with 0.3%w/w/trityl alcohol content. If the assay result was not ≤0.5% w/w tritylalcohol, the aqueous phase was charged with TBME (5.0 vol, 3.7 wt) andstirred at 25 to 30° C. for 10 to 20 min then repeated the separation.The combined aqueous solution was adjusted to 18 to 23° C., charged withpre-treated Ambersep 900 (OH form) resin (5/6^(ths) of the bulk treatedmaterial) and stirred for 15 min to check the pH. If the pH was <8.0,more Ambersep 900 resin (OH form) was added and stirred the solution for30 to 45 min at 18 to 23° C. The slurry was filtered and washed withwater for injection (2×4.0 vol) for 15 to 30 min per wash. The resinfilter cake on the filter washed with water for injection (3×4.0 vol)further for 15 to 30 min per wash until a result of ≤1.0% was obtainedby HPLC assay in each wash. The mother liquors and any wash obtainedcontaining ≥1.0% were clarified via a 1 m filter. The solution washeated to 40 to 45° C. and concentrated to 1.5 vol under reducedpressure at 40 to 45° C. After cooling the aqueous solution to 18 to 23°C. over 2 to 3 h and the mixture was aged for 60 min and charged withacetonitrile (9.5 vol) maintaining 18 to 23° C. at an approximatelyconstant rate over 1.5 to 2 h. The slurry was aged at 18 to 23° C. for 2h and cooled at 0 to 5° C. for 90 min. The solid was filtered through 20m cloth and washed with MeCN/water (5:1, 1.5 vol) and dried under an airatmosphere until MeCN content is <400 ppm by GC. The process yielded8.20 kg (99.83% purity) net weight of RX-3117 monohydrate,4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one1H₂0, (29.8 mol, 99.83% purity), equivalent to 66% total and 34% w/wyield.

FIG. 29 is a ¹H NMR showing RX-3117 made using the process described inExample 9. ¹H-NMR (400 MHz, DMSOd₆), δ 7.40 ppm, (d, J=7.3 Hz, 1H) CHcytosine, δ 7.20 ppm, (broad d, J=9.1 Hz, 2H) NH₂, δ 5.74 ppm, (d, J=7.3Hz, 1H) CH cytosine, δ 5.30 ppm, broad s, 1H, CH, δ 5.15 ppm, (d, J=7.1Hz, 1H) (OH), δ 5.00 ppm, (d, J=6.1 Hz, 1H) (OH), δ 4.80 ppm, (q, J=5.3Hz, 1H)(OH), δ 4.48 ppm, (q, J=5.3 Hz, 1H) CH, δ 4.17 ppm, (dd, J=9.1Hz, 3.8 Hz, 1H) CH, δ 4.13 ppm, (dt, J=6.1 Hz, 5.8 Hz, 1H) CH, δ 3.91ppm, (broad d, J=12.9 Hz, 2.8 Hz, 1H) CH

FIG. 30 is a ¹³C NMR of RX-3117 made using the process described inExample 9.

FIG. 31 is a ¹⁹F NMR of RX-3117 made using the process described inExample 9.

FIG. 32 is a Mass Spectrum of RX-3117 made using the process describedin Example 9. The mass spectrum was done using the ES+ filter that showsthe protonated species of RX-3117 (M+H) as well as an RX-3117 plussodium adduct (M+Sodium) at m/z=280.0 (a common species seen during thisanalysis method). The sodium comes from the analysis method, not themanufacturing process.

FIG. 33 is a Mass Spectrum of RX-3117 made using the process describedin Example 9. The mass spectrum was done using the ES− filter that showsthe M-H species of RX-3117 during the analysis process. The ES− and ES+filter methods together provide complete mass spectrum evidence forRX-3117.

In order to verify crystalline properties of material prepared accordingto the large scale synthetic process above, a microscopic comparison andwas made to crystals prepared by a laboratory scale high purity process,as well as a comparison of the X-Ray Powder Diffraction patterns. FIG.34 is a microscopic comparison of RX-3117 made according to the processof Example 9 (Top row) and prepared using a laboratory scale process(bottom row) under plain polarized light (left column) and crosspolarised light (right column). FIG. 35 is an X-Ray Powder Diffractiondata comparing RX-3117 made using a laboratory scale (top spectrum) andRX-3117 made using the process described in Example 9 (bottom spectrum).As can be seen, there is no significant difference in the crystalstructures.

It will be apparent to those skilled in the art that specificembodiments of the disclosed subject matter may be directed to one ormore of the above- and below-indicated embodiments in any combination.

While the invention has been disclosed in some detail by way ofillustration and example, it is apparent to those skilled in the artthat changes may be made and equivalents may be substituted withoutdeparting from the true spirit and scope of the invention. Therefore,the description and examples should not be construed as limiting thescope of the invention.

All references, publications, patents, and patent applications disclosedherein are hereby incorporated by reference in their entirety.

1. A process for preparing of4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one1H₂O (RX-3117-MH), comprising convertingtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11) to4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) in a continuous process with more than one step withoutisolation of any intermediate.
 2. The process of claim 1, furthercomprising the steps of: dissolvingtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11) in 2-methyl tetrahydrofuran; adding tetra-n-butylammoniumfluoride to form((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol)(INT12) in a reaction solution; and recovering INT12 in an organicphase.
 3. The process of claim 2, wherein said recovering INT12 in theorganic phase comprises the steps of: washing the reaction solution withan aqueous solution; separating an aqueous extraction from the organicphase having INT12; washing the aqueous extraction with 2-methyltetrahydrofuran to extract INT12 from the aqueous extraction; andcombining the extracted INT12 with the organic phase having INT12. 4.The process of claim 2, further comprising: adding triethylamine andmethanesulphonyl chloride in 2-methyl tetrahydrofuran to the INT12 inthe organic phase to form((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13) in a second reaction solution; and recoveringINT13 in DMSO.
 5. The process of claim 4, wherein said recovering INT13in DMSO comprises the steps of: adding the DMSO to the second reactionsolution with INT13; and removing at least 90% w/w of 2-methyltetrahydrofuran by distillation.
 6. The process of claim 4, furthercomprising: adding 2.5 equivalents of cesium carbonate and cytosine tothe INT13 in DMSO to form 4-amino-1-((3 aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) in a third reaction solution.
 7. The process of claim 6, furthercomprising: maintaining reaction temperature at about 33 to 37° C. 8.The process of claim 6, wherein the INT14 has a ratio of N- to O-isomersof over about 95:5.
 9. The process of claim 6, further comprising:adding an acid to the third reaction solution with INT14 to form4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one(RX-3117); washing the RX-3117 with methyl tert-butyl ether and water toform an organic phase and an aqueous phase having RX-3117; and purifyingthe RX-3117 to form RX-3117-MH.
 10. The process of claim 9, furthercomprising: charging the reaction mixture with methanol and distillingthe reaction mixture to remove acetonide until less than about 1.0% areaof the acetonide is detected prior to the washing step.
 11. The processof claim 9, wherein the washing step further comprises: separating theaqueous phase having RX-3117 from the organic phase; washing the aqueousphase having RX-3117 with methyl tert-butyl ether until less than about0.5% w/w trityl alcohol is detected in the aqueous phase; adding a basicanion resin to the aqueous phase having RX-3117 to form a slurry;filtering the slurry to retain a mother liquor; concentrating the motherliquor to form a concentrate; and adding acetonitrile to the concentrateto form purified RX-3117-MH.
 12. A continuous process for preparing4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14) fromtert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane(ASM11), comprising the steps of: dissolving the ASM11 in 2-methyltetrahydrofuran; adding tetra-n-butylammonium fluoride to form((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol)(INT12); adding trimethylamine and methanesulphonyl chloride in 2-methyltetrahydrofuran to the INT12 to form((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13); and adding cesium carbonate and cytosine tothe INT13 to form4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14); wherein the steps are performed in one or more fixed reactorswithout isolation of INT12 or INT13.
 13. A continuous process forpreparing4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one1H₂O (RX-3117-MH) from 4-amino-1-((3 aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one(INT14), comprising the steps of: reacting the INT14 with an acid toform 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one(RX-3117); washing the RX-3117 with methyl tert-butyl ether and water toform an organic phase and an aqueous phase having RX-3117; separatingthe aqueous phase having RX-3117 from the organic phase; washing theaqueous phase having RX-3117 with methyl tert-butyl ether until lessthan about 0.5% w/w trityl alcohol is detected in the aqueous phase;adding a strongly basic anion resin to the aqueous phase having RX-3117to form a slurry; filtering the slurry to retain a mother liquor;concentrating the mother liquor to form a concentrate; addingacetonitrile to the concentrate to form purified RX-3117-MH; andisolating the purified RX-3117-MH; wherein the steps are performed inone or more fixed reactors.
 14. A method of sensitizing a cell to anapoptotic signal comprising contacting the cell with an effective amountof a compound of formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.15. A method of treating one or more symptoms of cancer in a subject,comprising administering a compound of formula (I)

or a hydrate, a solvate, or a pharmaceutically acceptable salt thereofin an amount effective to inhibit methyltransferase and to upregulate atleast one hypomethylated target in the subject.